The tail of Listeria monocytogenes : Lessons learned from a bacterial pathogen (cont.)

1. 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

2. 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 loss addition Filament density Distance um from back

3. C term N term Signal peptide Proline-rich repeats Bact. Memb. anchor sequence 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

4. Which factors enhance actin polymerization ? C term N term Proline-rich repeats VASP Bact. Memb. anchor sequence Signal peptide 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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