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