the tail of listeria monocytogenes : lessons learned from a bacterial pathogen (cont.)
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The tail of Listeria monocytogenes : Lessons learned from a bacterial pathogen (cont.)
1. How do Listeria make tailsNucleation, growth
2. Role of ABPs in tail formation3. Other motile pathogens
How does actin polymerization drive the movement of Listeria?
• 1. “Insertional” actin polymerization occurs at back edge of bacterium– Polymerization fluorescently
labeled actin shows brighter regions at back edge
• 2. Photobleaching experiments show that the tail remains stationary as bacterium moves forward
• 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
Distance um from backFi
lam
ent d
ensi
ty
addition
loss
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
Bact. Memb. anchor sequence
N term C term
Signal peptide Proline-rich repeats
• 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
Which factors enhance actin polymerization ?
Bact. Memb. anchor sequence
N term C term
Signal peptide
Proline-rich repeats
VASP
P
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., 2000JCS 113 p.1415
• 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
ARP2/3 nucleates actin filament growth in Listeria
• “Y” shaped cross-links containing ARP2/3 are present
• Evidence of other kinds of crosslinking exists
Organization of actin filaments in Listeria is similar to that of
lamellipodia
• 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
Capping and severing ABPs are found in Listeria tails
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 formationby 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 0.1M 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 0.5M
• 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
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