Investigation of Curli-Specific Proteins CsgD and CsgA Reveals a Potential Target for Neurodegenerative Therapeutics and Provides Structural Insights into the Bacterial Amyloid CsgA

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Jude Kinkead (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: A growing body of evidence implicates curli-containing biofilms in the onset of neurodegenerative pathologies like Alzheimer’s and Parkinson’s diseases. Curli-containing biofilms are composed primarily of two bacterial amyloid proteins, CsgA and CsgB. Amyloid proteins are typically intrinsically disordered or contain intrinsically disordered regions. As a result, their native state is difficult to define due to diverse conformational sampling. In addition, these proteins are prone to misfolding and common secondary structural features provide an avenue for template-driven polymerization that results in an insoluble protein deposit rich in ß-sheets. \n We are host to an array of microorganisms, which are able to directly interact with their hosts through the gut-brain axis. The vagus nerve provides a physical connection between the gut and brain, allowing for bidirectional communication. Curli-containing biofilms produced by enteric gut bacteria like E. coli and S. Typhimurium have been shown to promote motor impairment in mice, as well promote motor impairment and aSyn aggregation in aged Fischer 344 rats and C. elegans. In addition, the bacterial amyloid CsgA has been shown to accelerate aSyn in vitro as well as colocalize with aSyn in C. elegans and human neuroblastoma cells. Soluble oligomers of CsgA may be able to serve as a template for rapid polymerization of aSyn through a process known as cross-seeding. In addition, curli fibrils are recognized by TLR2 and may contribute to chronic low-grade inflammation characteristic of these disorders. \n Expression of curli fibrils is dependent upon a number of transcription factors, as well as the DnaK-DnaJ-GrpE chaperone system. Following translation of the csgDEFG-encoded proteins, the master-regulator of biofilm development, CsgD, upregulates the transcription of the csgBAC operon. CsgA and CsgB are kept soluble within the cell by CsgC and DnaK prior to periplasmic secretion through SecYEG machinery. CsgA and CsgB are kept soluble by CsgE and transported through the pore complex CsgG. As CsgA and CsgB exit the cell, they are postulated to begin adopting the ß-sheet rich structure similar to the mature fibril. \n A class of compounds have shown specificity for response regulators and have demonstrated efficacy not only in inhibiting biofilm formation, but also dispersing established biofilms. The 2-aminoimidazole compounds, initially discovered in the Agelasidae family of marine sponges, contain a 2-aminoimidazole pharmacore linked to various substituent groups. The 2-aminoimidazole pharmacore is predicted to bind an electronegative pocket separating the receiver and DNA-binding domains of a response regulator, while the substituent group maintains contacts with both domains. Precise tuning of the substituent group can provide for drastically increased specificity to a response regulator. In this work, a variety of expression and purification approaches were implemented to isolate both E. coli CsgD and S.Typhimurium CsgD. Following purification, these proteins were screened utilizing a thermal shift assay to investigate their propensity for ligand binding by the 2-aminoimidazole compounds. The N-terminal domain of E. coli CsgD, as well as the N-terminal domain and full-length constructs of S. Typhimurium CsgD suggest ligand binding in samples treated with AGL-869. \n While there are no known cures for either Alzheimer’s or Parkinson’s disease, efforts to develop effective diagnostic methods have identified a target group. This group is the group of stable oligomeric intermediates adopted by endogenous amyloids. These oligomeric intermediates are thought to be capable of exerting cytotoxic effects via formation of a pore-like complex with aberrant function. \n As a result of their propensity for aggregation, a typical approach utilized for the isolation of soluble, monomeric amyloids is the application of high concentrations of denaturants like guanidinium hydrochloride. While this is a standard approach, its application to an intrinsically disordered protein known to interact with chaperone complexes may prohibit the purified isolate from inhabiting its native range of conformations. This could potentially limit investigations of stable oligomeric species thought to be responsible for cytotoxic effects. In order to combat this, we co-expressed the DnaK-DnaJ-GrpE in order to efficiently solubilize and isolate recombinant E. coli CsgA under native conditions. Natively-purified CsgA was shown to exhibit secondary structural characteristics that diverge from established literature. Characterization of natively-purified CsgA and initial comparisons to CsgA purified using high concentrations of denaturant are also included.

Additional Information

Publication

Thesis

Language: English

Date: 2023

Subjects

Amyloid;Alzheimer's;Parkinson's;Curli;Response Regulators

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