Summary idea led me to this study!

1. AMYLOID OLIGOMERS
AND FUNCTIONAL AMYLOIDS
LE THI KHANH NGHIA
Student ID: 2017310896
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2. ALZHEIMER’S DISEASE (AD)
PROTEIN-MISFOLDING DISEASES
FUNCTIONAL AMYLOIDS
FROM BACTERIA TO HUMAN
THE FIRST STEPS
FOR REPRESENTATIVE AMYLOID SIMULATION
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3. • Prevalence and incidence
ALZHEIMER’S DISEASE (AD)
- By 2005:
+ 24.6 million people had dementia
+ 4.6 million new cases arising annually
+ ~70% of these cases were attributed to AD
- China and Western Pacific
exhibited the highest prevalence of
dementia. Follwed by Western
Europe and North America.
- The incidence rate for dementia
increased exponentially with
age, with the most notable rise
occurring through the seventh
and eighth
=> The prevalence and incidence
rates for AD also increase
exponentially with age.
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4. - Mechanism causing AD
ALZHEIMER’S DISEASE (AD)
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5. PROTEIN-MISFOLDING DISEASES
Mechanism
???
A representative simulation
of amyloid for studying aggregation mechanism
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6. FUNCTIONAL AMYLOIDS FROM BACTERIA TO HUMAN
• E. coli is the most understandable bacteria
• Produce protein in the short-term
• Biochemical and structural properties as
eukaryotic amyloid fibers
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7. THE FIRST STEPS FOR REPRESENTATIVE AMYLOID SIMULATION
CURLI
• The major proteinaceous component of
complex extracellular matrix produced by
Enterobacteriaceae, involved in adhesion
to surfaces, cell aggregation, and biofilm
formation.
• A part of a growing number functional
amyloid with biochemical and structural
properties as eukaryotic amyloid fibers.
(nonbraching and β-sheet rich fibers)
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8. Congo Red (CR) Assays to Assess Curli production
* CR assay on YESCA CR Agar
* Post-staining CR assay
• Wild-type curli-producing
stain red.
• Curli defective mutants
are usually pink or white.
• E. coli mutants with hyper
curli production
sometimes stain dark red.
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9. 9
Curli biogenesis
Operon Composition Function of gene
CsgA 30% of
sequence
identify
Major structural subunit
CsgB Nucleator protein
CsgD Center of the curli regulatory network
CsgG Required for stability and secretion of CsgA, CsgB
CsgE, CsgF
Interact with CsgG, required for efficient curli
assembly
- Genes impotant
for curli formation
in E. Coli.

10. Purification of Curli subunits
Method Non-denaturing Denaturing
• Advantage
o Avoid denaturation of target
protein
o Less time-consuming
o Cell can be stored in a
long term
• Disadvantage
o Time-consuming
o Supertanant cannot be
stored > 1 day
o Low yield
• Stimulation
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11. Purification of Curli subunits
* Non-denaturing Purification of CsgA or truncated CsgB
• Expression strain: LSR12/pMC1/pMC3
• This strain resistant to Ampicilin (Amp) and Chloramphenicol (Cm)
• CsgA (truncated CsgB) can be collected by Affinity Chromatography
LSR12
CsgDEFG csgBA
deletion mutation
in E. coli C600
pMC1
cloning csgG into the NcoI and BamHI
sites of pTrc99A plasmid
pMC3
cloning full-length C-terminal His6-
tagged CsgA into the NdeI and EcoRI
sites of pHL3 plasmid.
• Protein experession is induced with IPTG
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12. Purification of Curli subunits
* Denaturing Purification of CsgA or CsgB (bacterial lysate)
• Expression strain: NEB C2566/pNH11 (CsgA)
NEB C2566/pAN1 (CsgB)
• This strain resistant to Ampicilin (Amp) and Chloramphenicol (Cm)
• Use GdnHCl to lyse the cell
• Collect protein by Affinity Chromatography
NEB C2566: T7 expression strain of E. coli
pAN1
plasmid made by cloning C-
terminal His6-tagged CsgB
without Sec signal sequence
into pET11d vector
pNH11
plasmid made by
cloning C-terminal
His6-tagged CsgA
without Sec signal
sequence into pET11d
vector
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13. Benefits of Curli subunits
Purified
Subunits
Mechanism of aggregation
Biofilm formation
Function for adhesion
(surface interaction)
Polymerization, programmable biofilm etc
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15. THANKS
FOR YOUR ATTENTION!
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16. REFERENCES
• Dobson, Christopher M. "The Amyloid Phenomenon and Its Links with
Human Disease." Cold Spring Harbor Perspectives in Biology (2017):
a023648.
• Fowler, Douglas M., et al. "Functional amyloid–from bacteria to
humans." Trends in biochemical sciences 32.5 (2007): 217-224.
• Zhou, Yizhou, et al. "Experimental manipulation of the microbial
functional amyloid called curli." Bacterial Cell Surfaces: Methods and
Protocols (2013): 53-75.
• Reitz, Christiane, Carol Brayne, and Richard Mayeux. "Epidemiology of
Alzheimer disease." Nature Reviews Neurology 7.3 (2011): 137-152.
• Barnhart, Michelle M., and Matthew R. Chapman. "Curli biogenesis and
function." Annu. Rev. Microbiol. 60 (2006): 131-147.
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