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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.

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The human body is a dangerous place for a bacteria to be!

Antibodies

Neutrophils

Complement

Innate response - lysozymes



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Some bacteria gain entry to cells by forcing them to phagocytose them.

Bacteria secrete

Factors that stimulate

Macrophagocytosis

Once engulfed

Bacteria digest the

phagolysosome

Now cells can grow

Within the cell


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Fibroblast protrusion, phagocytose them.Louise CramerUniversity College London


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GFP-actin. phagocytose them.

Stimulated

Macropinocytosis


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Killing phagocytosis phagocytose them.

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 elaborate adhesion (Pedestal), that prevents phagocytosis and removal by flushing

Escherichia

Yersinia

Shigella

Staphylococcus

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 elaborate adhesion (Pedestal), that prevents phagocytosis and removal by flushing 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 elaborate adhesion (Pedestal), that prevents phagocytosis and removal by flushing

Dirty Rat Homo sapiens

Dirty Rat Rattus norvegicus


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WHO reports 1,000 to 3,000 cases of plague every year! elaborate adhesion (Pedestal), that prevents phagocytosis and removal by flushing


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Other elaborate adhesion (Pedestal), that prevents phagocytosis and removal by flushingYersinia 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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Type III activities

Type II

Type I

Outer membrane

Periplasm

Inner membrane

A

A

A

The three main types of bacterial secretion

Type III is most often associated with pathogenic bacteria


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The most common pathogenic activitiesE.coli

Abbrev. Full name Common name and features inocolum Source

ETEC Enterotoxin E.coli Montizuma’s revenge, traveller’s 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 Enterohaemorrhagic (O157)“Hamburger disease” Shiga toxin 3 Cattle faeces, meat


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Interactions of the common pathogenic activitiesE.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 filaments (a), then through intimin (b).. 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 filaments (a), then through intimin (b).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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Cell death & loss filaments (a), then through intimin (b).

Lumen of gut

Movement of fresh cells up from crypt

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.


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Microfilaments are present in filaments (a), then through intimin (b).

‘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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O contact and microvilli

II

+

Actin

NH-C-CH

I

H-S-H

C-

2

2

Actin

NH-C-CH

-S-H

C-

2

2

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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Wiskott-Aldrich Syndrome Protein (WASP) contact and microvilli

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The Arp2/3 complex contact and microvilli

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 contact and microvilli

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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Edwina Currie components

Eggs

Salmonella

Salmonella


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Commensal componentsSalmonella calm the Immune system


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Pathogenic componentsSalmonella disrupt normal cell function

Salmonella

Type III secretion

Rac

GDP

Cdc42

GDP

SopE

SopB

SipC

SipB

SptP

SipA

RacGTP

Cdc42

GTP

Plastin

Active WASP

Actin polymerizing

and bundling

Arp2/3 complex

Inactive WASP


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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


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Pathogenic invasion.Salmonella disrupt normal cell function

Salmonella

Type III secretion

Rac

GDP

Cdc42

GDP

SopE

SopB

SipC

SipB

SptP

SipA

RacGTP

Cdc42

GTP

Plastin

Active WASP

Actin polymerizing

and bundling

Arp2/3 complex

Inactive WASP


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The invasion.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 can’t 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 invasion.

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 invasion.

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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Actin+SipC-C 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

Actin+SipC-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 (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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