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 Distance um from back Filament density addition loss

3. 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

4. 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

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 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

8. “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

9. 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

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 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

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