Download Making Attachments: Curli Production in Bacterial Communities

Making Attachments:
Curli Production in
Bacterial Communities
Deborah Yager
Stanford IISME 2010
Lynette Cegelski, Mentor
Funded by Terman Fellowship Award
Figure 1C4: MC4100 E.Coli deep-etched on glass before rotary shadowing with Pt showing curli fibrils
Outline
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What is a biofilm?
Biofilms and everyday life
Curli
Basic model for curli protein interactions
More details on each protein subunit
Congo red binding assay
Experimental design
What is a Biofilm?
Biofilms and the Environment
Biofilm Life Cycle
The biofilm life cycle in three steps:
attachment, growth of colonies (development),
and periodic detachment of planktonic cells.
Bioflims and Disease
Common sites of biofilm infection.
Once a biofilm reaches the
bloodstream it can spread to any
moist surface of the human body.
Microbes that colonize vaginal
tissue and tampon fibers can
become pathogenic, causing
inflammation and disease
such as Toxic Shock
Syndrome.
Bioflims and Disease
Why Bacteria Make Attachments
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Bacteria prefer to attach to surfaces rather than
live in suspension
Advantages to attachment:
1) higher nutrient concentration (carbon source)
2) protection from hostile environments
3) formation of biofilms, “a structured community
of bacterial cells enclosed in a self-produced
polymeric matrix and adherent to an inert or
living surface.”
Advantages of Biofilms
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In a biofilm, energy required to find food is shared
Biofilm members on the edge are first line of
defense
Bacteria in biofilms secrete a protective matrix of
polysaccharides and proteins (i.e. cellulose & pili)
One set of secreted
proteins form curli fibers
Biofilm in the gut
Curli as Amyloid
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Amyloid is an extracellular structure built of protein
subunits in a cross-beta sheet formation
Cross beta
sheet
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Amyloid aggregates are implicated in several
diseases, including Alzheimer’s and Parkinson’s
Curli are adhesive extracellular functional amyloid
fibers secreted by bacteria
Curli Production
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Curli are secreted by Gram negative bacteria
including Escherichia Coli (E. coli) and Salmonella.
Together with cellulose and other extracellular
matrix components, curli help bacteria attach to
surfaces and stick together to form a biofilm.
While curli form spontaneously in vitro (in a test
tube with no cells), in vivo (in life) curli production
requires specialized molecular machinery.
Curli Molecular Machinery
Genetics Nomenclature
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csgA means “gene that codes for protein A”
CsgA means “Protein A coded by curli
specific gene (csg)”
Practice:
 What does CsgG mean?
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What does csgG mean?
Quick Guide to Curli Proteins
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CsgA: major curli subunit, forms fibers
CsgB: minor curli subunit, nucleation
CsgD: transcriptional activator
CsgE: gatekeeper or plug
CsgF: chaperone protein
CsgG: outer membrane pore
CsgA
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Major fiber subunit
13 kDa w/o Sec sequence
Amyloidogenic imperfect
repeating units (R1-5)
Gln/Asn-rich repeat motif
3 domains
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N-terminal Sec sequence
(2 kDa)
First 22 amino acids
C-terminal Amyloid core
Fibers rich in -sheet
secondary structure
CsgA (cont.)
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Polymerization responds to self-seeding and
CsgB nucleation
Proposed model: R1 interacts with CsgB, R5
interacts with R1 of next CsgA
A
A
A
A
B
B
F
E
Congo Red Binding Assay
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Congo red is an amyloid dye that binds to CsgA
subunits when they are polymerized in a fiber
Bacterial cells grown on nutritional media with
Congo red will stain red if curli fibers are present
CR
Congo Red Binding Assay
Curli -
Curli +
Interbacterial Complementation
Interbacterial Complementation
Interbacterial Complementation (IC):
• recipient strain is streaked first (down)
• donor strain streaked next (across)
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Congo red dye binds to
polymerized CsgA
∆A mutant = no fibers,
cells white on CR plate
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Figure 3D4: Donor CsgA+ CsgB-, Recipient CsgA- CsgB+
Can accept CsgA in IC
CsgB
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Figure 1A2
Minor fiber subunit
17 kDa (whole)
5 repeating units
Gln/Asn-rich repeat
motif
~30% sequence
identity with CsgA
R1-R4 amyloidogenic
A
CsgB (cont.)
A
A
B
B
A
F
E
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Nucleates CsgA
WT CsgB cell-associated
∆B mutant = no fibers,
cells white on CR plate
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Secretes soluble CsgA
outside the cell
Can donate CsgA in IC
CsgD
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5 kDa (whole)
Transcriptional regulator for curli genes
Critical activator of cellulose biosynthesis
pathway
∆D mutant = no curli or cellulose
Represses genes inhibiting biofilm formation
in E. coli
CsgE
A
A
A
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A
B
B
F
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E
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15 kDa (whole)
Interacts with CsgG at
outer membrane
Gatekeeper for CsgG pore
∆E mutant: very few fibers
(pale cells, CR-), arrange
into rings
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Figure 3D4: Donor CsgA+ CsgB-, Acceptor CsgA- CsgB+
Cannot donate CsgA
Can accept and form fibers
Suggests CsgE needed for
CsgA polymerization stability
CsgF
A
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A
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A
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A
B
B
F
E
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Chaperone-like, helps CsgB
nucleation
15 kDa (whole)
Secreted across outer
membrane, interacts with CsgG
Degraded with increasing
concentration of proteinase K,
suggests CsgF exposed on cell
surface
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Same observed in ∆A, ∆B, ∆E
mutants, localization not
dependent on A, B, or E
CsgF (cont.)
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∆F mutant = no cellassociated fibers, CsgA
secreted soluble
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CR: Scraped whole cells
appear white, suggests
fibers mislocalized to agar
Figure 3A/B5
CsgG
A
A
A
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A
B
B
Outer membrane pore
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F
E
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30.5 kDa (whole)
∆G mutant = no fibers
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Figure 1A6
Stabilizes curli subunit
proteins
12-15 nm across with 2 nm
pore
CsgA and CsgB not secreted
to cell surface, not in
periplasmic space
Can neither donate nor
accept in IC
Congo Red Binding Assay
Curli -
Curli +
Experimental Design
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Single variable of interest: want to minimize
influence of other variables
Controlled experiment uses scientific controls:
positive control and negative control
Positive control confirms that your methods
can produce a positive result
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minimizes false negatives
Negative control confirms that your methods
do not produce an unrelated effect
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minimizes false positives
demonstrates baseline result obtained without
positive result (“background” value)
Our Experiment
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Experimental variable: a single curli-specific gene
(csg) mutation (MC4100DcsgA)
Positive control: wild type MC4100 cells
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shows a positive result with our methods (Congo red
binding)
Negative control: cells missing complete csg
sequence (MC4100Dcsg)
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shows “background” (negative) result obtained when
no curli genes are expressed
Curli mutant
UTI89
UTI89
MC4100
References
1. Cegelski Lab. “Structure, Function, and Disruption of Microbial Amyloid Assembly and Biofilm
Formation.” 2009.
2. Hammer, N.D.; Schmidt, J.C.; Chapman, M.R. The curli nucleator protein, CsgB, contains an
amyloidogenic domain that directs CsgA polymerization. PNAS 2007, 104, 12494-12499.
3. Cegelski, L.; Pinkner, J.S.; Hammer, N.D.; Cusumano, C.K.; Hung, C.S.; Chorell, E.; Aberg, V.; Walker,
J.N.; Seed, P.C.; Almqvist, F.; Chapman, M.R.; Hultgren, S.J. Small-molecule inhibitors target
Escherichia coli amyloid biogenesis and biofilm formation. Nature Chemical Biology 2009, 12, 913-919.
4. Chapman, M.R.; Robinson, L.S.; Pinkner, J.S.; Roth, R.; Heuser, J.; Hammar, M.; Normark, S.; Hultgren,
S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 2002, 295, 851855.
5. Nenninger, A.A.; Robinson, L.S.; Hultgren, S.J. Localized and efficient curli nucleation requires the
chaperone-like amyloid assembly protein CsgF. PNAS 2009, 106, 900-905.
6. Robinson, L.S.; Ashman, E.M.; Hultgren, S.J.; Chapman, M.R. Secretion of curli fibre subunits is mediated
by the outer membrane-localized CsgG protein. Molecular Microbiology 2006, 59, 870-881.
7. Wang, X.; Chapman, M.R. Curli provide the template for understanding controlled amyloid propagation.
Prion 2008, 2, 57-60.
8. Wang, X.; Zhou, W.; Ren, J.; Hammer, N.D.; Chapman, M.R. Gatekeeper residues in the major curlin
subunit modulate bacterial amyloid fiber biogenesis. PNAS 2010, 107, 163-168.
9. Ashman, E.; Chapman, M.R. Polymerizing the fibre between bacteria and host cells: the biogenesis of
functional amyloid fibres. Cellular Microbiology 2008, 1-8.
10. Zakikhany, K.; Harrington, C.R.; Nimtz, M.; Hinton, J.C.D.; Romling, U. Unphosphorylated CsgD
controls biofilm formation in Salmonella enterica serovar Typhimurium. Molecular Microbiology 2010,
accepted article.
11. Ashman Epstein, A.; Reizian, M.A.; Chapman, M.R. Spatial clustering of the curlin secretion lipoprotein
requires curli fiber assembly. Journal of Bacteriology 2009, 191, 608-615.