| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Chorell, Erik, et al.
(författare)
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Bacterial Chaperones CsgE and CsgC Differentially Modulate Human α-Synuclein Amyloid Formation via Transient Contacts.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 10:10, s. e0140194-
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson's disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.
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| 3. |
- Jain, Neha, et al.
(författare)
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Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:46, s. 12184-12189
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Tidskriftsartikel (refereegranskat)abstract
- During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer’s-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.
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| 4. |
- Sampson, Timothy R., et al.
(författare)
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A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice
- 2020
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Ingår i: eLife. - 2050-084X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Amyloids are a class of protein with unique self-aggregation properties, and their aberrant accumulation can lead to cellular dysfunctions associated with neurodegenerative diseases. While genetic and environmental factors can influence amyloid formation, molecular triggers and/or facilitators are not well defined. Growing evidence suggests that non-identical amyloid proteins may accelerate reciprocal amyloid aggregation in a prion-like fashion. While humans encode ~30 amyloidogenic proteins, the gut microbiome also produces functional amyloids. For example, curli are cell surface amyloid proteins abundantly expressed by certain gut bacteria. In mice overexpressing the human amyloid α-synuclein (αSyn), we reveal that colonization with curli-producing Escherichia coli promotes αSyn pathology in the gut and the brain. Curli expression is required for E. coli to exacerbate αSyn-induced behavioral deficits, including intestinal and motor impairments. Purified curli subunits accelerate αSyn aggregation in biochemical assays, while oral treatment of mice with a gut-restricted amyloid inhibitor prevents curli-mediated acceleration of pathology and behavioral abnormalities. We propose that exposure to microbial amyloids in the gastrointestinal tract can accelerate αSyn aggregation and disease in the gut and the brain.
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