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Bacteria and the cytoskeleton
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The human body is a dangerous place for a
bacteria to be! Antibodies Neutrophils Compl
ement Innate response - lysozymes
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Many bacteria find it much more comfortable
inside the cells of its host.
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Some bacteria gain entry to cells by forcing them
to phagocytose them.
Bacteria secrete Factors that stimulate Macrophago
cytosis
Once engulfed Bacteria digest the phagolysosome
Now cells can grow Within the cell
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Fibroblast protrusion, Louise Cramer University
College London
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GFP-actin. Stimulated Macropinocytosis
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Killing phagocytosis tight compartment
Stimulated phagocytosis loose compartment
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Other bacteria (EPEC) stimulate the production of
an elaborate adhesion (Pedestal), that prevents
phagocytosis and removal by flushing
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Salmonella Escherichia Yersinia Shigella Staph
ylococcus Legionella Listeria
Many bacteria subvert normal cytoskeletal
function in order to parasitize their eukaryotic
host through either adhesive complexes or
inducing macro-pinocytosis (Most nasty
bacteria are named After people!!)
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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
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Xenopsylla cheopis
Dirty Rat Homo sapiens
Dirty Rat Rattus norvegicus
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WHO reports 1,000 to 3,000 cases of plague every
year!
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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.
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Bacteria inject toxins into cells to subvert
their activities
The hypodermic syringes that they use are
modified flagella
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The three main types of bacterial secretion Type
III is most often associated with pathogenic
bacteria
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The most common pathogenic E.coli
Abbrev. Full name Common name and features
inocolum Source ETEC Enterotoxin
E.coli Montizumas revenge, travellers tummy
108 Faecal usually comparatively mild,
(Diacalm grade) contamination EIEC Enteroinvasive
Invades, Shigella pathogenicity island high
Food waterborne EPEC Enteropathogenic
Pedestal formation, infant diarrhoea 108 - 10
Nosocomial community EHEC Enteroha
emorrhagic (O157)Hamburger disease Shiga
toxin 3 Cattle faeces, meat
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Interactions of the common pathogenic E.coli with
epithelial cells
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Interaction of EPEC with epithelium first through
EspA filaments (a), then through intimin
(b). Knutton et al, 1998. Nucleolin is a third
binding site.
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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.
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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.
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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.
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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
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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
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Pyrene-actin method to measure polymerisation
kinetics
Pyrene Excited
Light emitted at
at 366nm
384nm measured
Pyrene-actin in quartz cuvette
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Actin-Binding Proteins modify actin polymerization
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Wiskott-Aldrich Syndrome Protein (WASP)
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The Arp2/3 complex An actin-binding group of
proteins pivotally involved in the regulation of
actin polymerisation.
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Analysis of the WASP domains required for
Pedestal formation
WASP-WT
WASP-DC
WASP-DGBD
Kalman et al, 1999 Nature Cell Biol. 1 389-391.
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Pedestal formation and localization of Arp2/3
complex components.
Kalman et al, 1999 Nature Cell Biol. 1 389-391.
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Pedestal formation by EPEC
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Salmonella
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Commensal Salmonella calm the Immune system
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Pathogenic Salmonella disrupt normal cell function
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Hints of Plastins 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
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Pathogenic Salmonella disrupt normal cell function
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The Salmonella cycle of infection
SPI1
SPI2
Initial contact
A fresh actin wave of actin polymerization
results in the vacuole being covered in actin.
Actin polymerization and phagocytosis
Injection By type III secretion
Phagosome stimulates new protein
secretion Through a second type III machine
Lysosomes cant fuse
Nucleus
Some time later an actin ADP-ribosylating enzyme
disassembles the structure for unknown reasons
(cell lysis spread?).
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Gel filration of SipC
SipC and actin
SipC
Actin
SipC and actin (Higher power)
SipC and actin
Hayward, R.D. Koronakis, V. 1999 EMBO J. 18,
4926-4934.
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Actin SipC-C
Actin SipC-N
Gel filtration
Actin SipC-C
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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
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ActinSipC-C
Actin
ActinSipC-C Cyto
Actin Cyto
SipC C-terminal domain
SipC-C inhibition by Cytochalasin D
E.M. of actin with SipC-C and SipC-N
Bundle
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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 F-actin SipC (C), SipC-C (F).
Injected cells are indicated by arrows.
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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).
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The End
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