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Bacteria and the cytoskeleton
The human body is a dangerous place for a bacteria to be!
Antibodies
Neutrophils
Complement
Innate response - lysozymes
Many bacteria
find it much
more
comfortable
inside the cells
of its host.
Some bacteria gain entry to cells by forcing them to
phagocytose them.
Bacteria secrete
Once engulfed
Factors that stimulate Bacteria digest the
Macrophagocytosis
phagolysosome
Now cells can grow
Within the cell
Fibroblast protrusion, Louise Cramer University College London
GFP-actin.
Stimulated
Macropinocytosis
Killing phagocytosis
tight compartment
Stimulated phagocytosis
loose compartment
Other bacteria (EPEC) stimulate the production of an elaborate
adhesion (Pedestal), that prevents phagocytosis and removal by
flushing
Many bacteria subvert
normal cytoskeletal function
in order to parasitize their
eukaryotic host through
either adhesive complexes or
inducing macro-pinocytosis
Salmonella
Escherichia
Yersinia
Shigella
Staphylococcus
(Most nasty bacteria are named
After people!!)
Legionella
Listeria
Yersinia pestis was responsible
for the Great Plagues.
During the 6th century AD, the plague
ravaged the known world over a 50 year
period causing 100 million deaths. The "black
death" again devastated Europe during the
14th century over a 5 year period causing 25
million deaths (25% of the European
population). The bacterium was named after
Yersin who identified it as being the causative
agent of an outbreak of plague in Hong Kong
Xenopsylla cheopis
Dirty Rat Homo sapiens
Dirty Rat Rattus norvegicus
WHO reports 1,000 to 3,000 cases of plague every year!
Other Yersinia cause disease.
Yersinia enterocolitica
Typically, only a small number of human cases of Yersiniosis are
recognized. Symptoms are like that of appendicitis and out breaks
are often detected by a sudden increase in appendectomies in a
particular region.
The Center for Disease Control & Prevention estimates that about
17,000 cases occur each year in the United States.
Bacteria inject toxins into cells to subvert their activities
The hypodermic syringes that they use are modified flagella
Type I
Type III
Type II
Outer membrane
Periplasm
Inner membrane
A
A
A
The three main types of bacterial secretion
Type III is most often associated with pathogenic bacteria
The most common pathogenic E.coli
Abbrev.
ETEC
Full name
Enterotoxin E.coli
Common name and features
inocolum
Montizuma’s revenge, traveller’s tummy
108
usually comparatively mild, (Diacalm grade)
Invades, Shigella pathogenicity island
high
Pedestal formation, infant diarrhoea
108 - 10
EIEC
EPEC
Enteroinvasive
Enteropathogenic
EHEC
Enterohaemorrhagic (O157)“Hamburger disease” Shiga toxin
3
Source
Faecal
contamination
Food & waterborne
Nosocomial
community
Cattle faeces, meat
E.coli
Intimin/Tir complex
Actin bundle
Enteroxin delivery
A
ETEC
Pedestal formation
B
EPEC
Shiga toxin delivery
C
EIEC
D
EHEC
Interactions of the
common pathogenic
E.coli with epithelial
cells
Interaction of EPEC with epithelium first through EspA filaments (a), then through intimin (b).
Knutton et al, 1998. Nucleolin is a third binding site.
Scanning E.M. of EPEC and epithelium. EspA filaments appear to insert into cell
(arrows in A), possibly to deliver EspB. EspA may be part of the Type III secretion
pathway, it is needed for EspB delivery. Note the pedestals are all of equal length.
Some pathogenic E.coli (EPEC, EHEC) put down their own
“Welcome mat”
Tir (translocated intimin receptor) is injected into host by
Type III secretion
Tir binds to host a-actinin, talin and vinculin all components
of the focal contact.
Nucleolin is a bacterial binding site for EPEC.
Cell death & loss
Movement of fresh cells up from crypt
Lumen
of gut
Cell division
By targeting
nucleolin E.coli are
able to attach to the
cells that will exist
for the longest
time.
E.coli bind
dividing cells in the
crypt and stay
attached as the are
conveyed to the
tips.
Microfilaments are present in
‘stress fibres’ that are attached
to ‘Focal adhesions’. They are
also present as a gel under the
plasma-membrane esp. at the
leading edge
The pedestal has features in common with both the focal contact
and microvilli
Arp2/3
WASP
a-actinin
Vinculin
Villin
Ezrin
Pedestal base
Myosin II
tropomyosin
Pyrene-actin method to measure polymerisation kinetics
O
II
NH-C-CH2 I
+
H-S-H2C-Actin
NH-C-CH2 -S-H2 C-Actin
Pyrene Excited
at 366nm
Light emitted at
384nm measured
Pyrene-actin in quartz cuvette
Actin-Binding Proteins modify actin polymerization
Monomer
Dimer
Trimer
Multimer
Extent of Polymerization
(Fluorescence)
Actin & Nucleating activity
Actin alone
Actin & Sequestering activity
Time
Wiskott-Aldrich Syndrome Protein (WASP)
Proline-rich
WH2
Acidic
C
Inactive
CRIB
Proline-rich
P
WH2
P
Actin Actin
N
Basic
Acidic
C
Arp complex
Active
G
N
The Arp2/3 complex
An actin-binding group of proteins pivotally involved in the
regulation of actin polymerisation.
Zero length crosslink
Arp3
Arp2
Non-zero length crosslink
Yeast two hybrid screen
p18
p19
p14
p35
p40
Microfilament binding
Analysis of the WASP domains required for Pedestal formation
WASP-WT
Proline-rich
P
WH2
P
Acidic
Actin Actin
C
Arp complex
CRI
B
Ba s
ic
G
N
WASP-DC
Proline-rich
P
WH2
P
Actin Actin
CR
IB
G
Bas
ic
N
WASP-DGBD
Proline-rich
P
P
WH2
Actin Actin
Acidic
C
Arp complex
Kalman et al, 1999 Nature Cell Biol. 1; 389-391.
Pedestal formation and localization of Arp2/3 complex components.
Kalman et al, 1999 Nature Cell Biol. 1; 389-391.
Pedestal formation by EPEC
6
1
2
3
4
5
E.coli
Int im in
T ir
Chp
Arp2/3 com plex
WA SP
Actin polymerization
Salmonella
Edwina Currie
Eggs
Salmonella
Commensal Salmonella calm the Immune system
Pathogenic Salmonella disrupt normal cell function
Salmonella
Type III secretion
Rac
GDP
SipA
SipB
SipC
SopE
SopB
Rac
GTP
Plastin
Cdc42
GDP
SptP
Cdc42
GTP
Active WASP
Actin polymerizing
and bundling
Arp2/3 complex
Inactive WASP
Actin binding site 1
EF
EF
CH
Actin binding site 2
CH' CH
CH'
Plastin/Fimbrin Structure
Hints of Plastin’s involvement in signalling & Bacterial invasion.
1). BPB inhibition of plastin inhibits IP3 dependent Ca2+ increase in PMNs.
2). Plastin is itself regulated by Ca2+.
3). Phosphorylation of plastin at Ser5 by PKA results in integrin activation in
PMNs stimulated by Fc receptor ligation
Pathogenic Salmonella disrupt normal cell function
Salmonella
Type III secretion
Rac
GDP
SipA
SipB
SipC
SopE
SopB
Rac
GTP
Plastin
Cdc42
GDP
SptP
Cdc42
GTP
Active WASP
Actin polymerizing
and bundling
Arp2/3 complex
Inactive WASP
The Salmonella cycle of infection
SPI1
SPI2
Initial contact
Injection
By type III
secretion
Actin
polymerization
and phagocytosis
Phagosome stimulates
new protein secretion
Through a second type III
machine
A fresh actin wave of
actin polymerization
results in the vacuole
being covered in actin.
Lysosomes can’t fuse
Nucleus
Some time later an actin ADP-ribosylating
enzyme disassembles the structure for unknown
reasons (cell lysis & spread?).
Gel filration of SipC
SipC and actin
SipC
Actin
SipC and actin
Hayward, R.D. & Koronakis, V. 1999 EMBO J. 18, 4926-4934.
SipC and actin
(Higher power)
Actin + SipC-C
Gel filtration
Actin + SipC-N
Actin + SipC-C
Co-sedimentation of SipC N-terminus
with actin bundles. Sedimentation of
actin bundles from a mixture of SipC-N
and F-actin (both 5 µM), demonstrating
formation of an actin-SipC-N complex.
Supernatants (S) and pellets (P) after
centrifugation
Actin+SipC-C
Actin
Actin+SipC-C+ Cyto
Actin + Cyto
SipC C-terminal domain
E.M. of actin with
SipC-C and SipC-N
SipC-C inhibition by Cytochalasin D
Bundle
Induction of cytoskeletal rearrangements in vivo by SipC and SipC-C
microinjection. Cultured HeLa cells fixed 30 min after microinjection with purified
SipC (upper panels) or SipC-C (lower panels) (3 µM). Cells (DIC; A and D) were
stained with polyclonal antibody to SipC and FITC-conjugated anti-rabbit IgG
[SipC (B), SipC-C (E)] and with Texas Red-conjugated phalloidin to visualize Factin [SipC (C), SipC-C (F)]. Injected cells are indicated by arrows.
Co-injection of SipC-N with GST-GFP. Cultured HeLa cells (DIC; A and D)
fixed 20 min after microinjection with GST-GFP alone (upper panels) or mixed
with SipC-N (lower panels) (3 µM). GST-GFP was visualized directly [GST-GFP
alone (B); + SipC-N (E)] and F-actin stained with Texas Red-conjugated
phalloidin [GST-GFP alone (C); + SipC-N (F)]. Injected cells are indicated by
arrows (N = nuclear injection).