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Transcript
The tail of Listeria monocytogenes : Lessons
learned from a bacterial pathogen (cont.)
1. How do Listeria make tails
Nucleation, growth
2. Role of ABPs in tail formation
3. Other motile pathogens
How does actin polymerization drive the
movement of Listeria?
• 1. “Insertional” actin polymerization
occurs at back edge of bacterium
loss
– Polymerization fluorescently labeled
actin shows brighter regions at back
edge
• 3. Depolymerization occurs at the
same rate throughout the tail
– tail length is usually constant
– a decreasing gradient of filament
density exists from the front to rear
of the tail
– F-actin half life = 30 sec
addition
Filament density
• 2. Photobleaching experiments show
that the tail remains stationary as
bacterium moves forward
Distance um from back
ActA is sufficient for actin polymerization
• Listeria can still move is the presence of drugs that inhibit
protein synthesis
• In the early 90’s used a genetic screen in mutant Listeria that
could not form tails, and “normal” ones
• Found a single gene actA - encodes a bacterial surface protein
ActA
• Can induce tail formation in:
– Immotile Listeria, other bacteria, polystyrene beads
C term
N term
Signal peptide
Proline-rich repeats
Bact. Memb.
anchor sequence
Which factors enhance actin polymerization ?
N term
Signal peptide
Proline-rich repeats
VASP
C term
Bact. Memb.
anchor sequence
P
• ActA does not bind directly to actin
• Which factors localize at the back of Listeria but not in the tail?
– 1. Immunofluorescence studies found VASP (vasodilator-stimulated
phosphoprotein)
– 2. Profilin
• VASP binds to the proline-rich region of ActA and binds actin
– Discovered by looking for host cell factors that would bind to ActA
– Associated with F-actin and focal adhesions in lamellae
• Profilin binds VASP
How is polymerization enhanced?
• VASP and profilin accelerate filament elongation but are not nucleators
– Evidence: Actin clouds form in profilin depleted cytoplasmic extracts
– VASP-actin complexes have no nucleating activity
• Poly proline regions bind multiple VASP molecules
– Evidence: Bacterial speed is proportional to number of proline-rich repeats in
ActA
• VASP recruits profilin to the bacterial surface
GFP-profilin concentration at back edge is
proportional to speed
• Profilin accumulates
as speed increases
– (and vice versa)
• Only accumulates on
moving bacteria
Geese, et al., 2000
JCS 113 p.1415
ARP2/3 nucleates actin filament growth in Listeria
• Arp2/3 isolated by column chromatography from platelet cytoplasm (Welch
et al., 1997)
• Nucleation activity of Arp2/3 is greatly enhanced by ActA
– in eukaryotic cells and is essential for Listeria tail formation
• The amino-terminal domain of ActA is sufficient
Organization of actin filaments in Listeria
is similar to that of lamellipodia
•
•
“Y” shaped cross-links containing ARP2/3 are present
Evidence of other kinds of crosslinking exists
Capping and severing ABPs are found in Listeria tails
• Cap Z and gelsolin – (+end cappers) found throughout tail
– Limit growth of actin filaments
– Both ABPs are enriched at bacterial surface but ActA is thought to suppress their
activities here
• ADF/Cofilin - found throughout tail
– important for increasing actin filament turnover by 10-100 times compared with
in vitro
– Immunodepletion leads to formation of very long tails - actin turnover rate?
– Addition of excess decreases tail length – actin turnover rate?
• Crosslinking proteins -eg. Fimbrin, -actinin - found throughout tail,
structural role
– introduction of dominant negative fragment of -actinin stops bacteria
movement
Other motile pathogens
• Shigella – infects colon
epithelial cells, causes bacillary
dysentery
• Vaccinia virus of the poxvirus
family – e.g. variola virus
(small pox)
• Entry into cells and nucleation
of tail formation differs but
general principle is the same
Mechanisms of tail formation
by other pathogens
• Arp2/3 activation achieved
differently
– Listeria –ActA
– Shigella and Vaccinia – N-WASP
• Shigella – N-WASP is recruited by
IcsA
• Vaccinia – A36R recruits N-WASP
indirectly via Nck and WIP
– Requires phosphorylation of tyrosine
112 on A36R
Minimal requirements for Listeria rocketing
•
•
•
•
•
•
•
•
In physiological ionic strength buffer (pH 7.5) and F-actin 7.5 M
ARP2/3 0.1M and an activator - Act-A, N-WASp
Profilin 2.5 M
Gelsolin
Capping protein 0.05 M
ADF/Cofilin 5 M
X-linker (a-actinin) 0.25 M
VASP 0.5M
•
From: Loisel et al., 1999, Nature, 401, p.613
More motile pathogens
• Rickettsia – causes Rocky
Mountain spotted fever and
others
• Tails are different from Listeria,
Shigella, Vaccinia
• Composed of long actin filaments
• NOT nucleated by Arp2/3
• Movement is ~ x3 slower
• Actin filaments are x3 more
stable
Surfing pathogens
• EPEC –enteropathogenic
Escherichia coli
– Causes infantile diarrhoea
• Infects cells by inserting a bacterial
protein (intimin - Tir) into host cell
membrane
• Bacterium binds to Tir
• Phosphorylation of Tyr474 in the
cytoplasmic tail of Tir induces actin
polymerization – forms a pedestal
• Pedestal is dynamic – allowing
bacterium to surf
• Functional relevance unknown