Presented by: Cac Bui

1. Phosphorylation of Immunity-Related GTPases by a Toxoplasmagondii-Secreted Kinase Promotes Macrophage Survival and Virulence Presented by: Cac Bui

2. Toxoplasmagondii • A species of parasitic protozoa • Invade nucleated host cells and form a modified compartment, parasitophorous vacuole (PV) • PV allows safe replication and avoids clearance of host cells (phagocytes, monocytes/Mφ) • Acute infection is asymtomatic, flu-like symtoms. • Cause toxoplamosis in immuno-compromised patients and pregnant women, can become fatal.

3. Clearance of T. gondii • Infection by T. gondiistimulates recruitment of inflammatory monocytes to sites of inflammation. • Macrophages upregulate Immunity-related GTPases (IRGs) onto the surface of PV which rupture the vacuole, resulting in parasite degradation. • However, the virulent type I T. gondii strain can resist recruitment and avoid clearance by IRGs. • Type I strain virulent is mediated by the serine threonine (S/T) kinase ROP18.

4. ROP18 • During host-cell invasion, ROP18 is secreted into cytosol in small vescicles derived from apical secretory organelles (rhoptries). • ROP18 is targeted to the external surface of the parasite-containing vacuole, where its kinase activity is essential for virulence. • The mechanism of virulence remains unknown.

5. Hypothesis • ROP18 phosphorylates IRGs and block their ability to eliminate intracellular parasites

6. Does ROP18 mediate survival of parasite within inflammatory monocytes?

7. Figure 1: ROP18 mediates survival within Inflammatory monocytes • Parasite expansion after i.p injection in vivo

8. Figure 1: ROP18 mediates survival within Inflammatory Monocytes • Parasite expansion after i.p injection in vivo • ROP18 plays a role in survival and growth of parasites within peritoneal cells.

9. Recruitment of inflammatory monocytes to site of infection (peritoneum) • Flow cytometry & analysis

10. Recruitment of inflammatory monocytes to site of infection (peritoneum) • Flow cytometry & analysis • ROP18-expressed parasites expand rapidly within inflammatory monocytes

11. Parasitic clearance in elicited Gr1+monocytesin vitro

12. Parasitic clearance in elicited Gr1+monocytesin vitro • Parasite expressing active ROP18 resist clearance by Gr1+monocytes. • ROP18 allows parasite to establish a critical foothold within inflammatory monocytes early in infection

13. Figure 2: ROP18 mediates Protection of parasite-containing vacuole • Normal vs. Vesiculation of membrane, parasite degradation

14. How does ROP18-expressing parasite evade the clearance of IRGs?

15. Figure 3: ROP18 blocks Irgb6 Loading • Evaluate recruitment of IRGs to the parasite-containing vacuole by Irgb6 localization • Parasite expressing ROP18 blocks Irgb6 loading to PV Cryo-immuno-EM

16. How does ROP18 block IRGs loading?

17. Figure 4: ROP18 co-precipitates and phosphorylates IRGs • A-Immuno-precipitation of Igrb6 from macrophages • Interaction of Irgb6 with ROP18 • 3 fold increased in ROP18 D/A

18. Figure 4: ROP18 co-precipitates and phosphorylates IRGs • B- Phosphorylation of ROP18 and Irgb6 in a kinase reaction in vitro • IP Irgb6 & ROP18 separately • Incubation in hotkinase reaction • Phosphorylation of ROP18s • Irgb6 is detected only in active ROP18

19. Figure 4: ROP18 co-precipitates and phosphorylates IRGs • C- Phosphorylation of Irgb6 and ROP18 in vivo • 32P ortho-P labeled activated MΦ • Infect and IP with Irgb6 • * ROP18 phosphorylates Irgb6 in vivo

20. Figure 4: ROP18 co-precipitates and phosphorylates IRGs • Similar results with other IRGs , Irga6 & Irgb10 • ROP18 co-precipitates and phosphorylates IRGs

21. What’s the target reference of ROP18?

22. Figure 5: ROP18 targets a conserved motif in SWI of IRGs Sequence-based WebLogo Positional-scanning peptide array • ROP18 show strong preference for Thr over Ser, and for Thr at multiple positions • Slight preference for acidic residues upstream and bulky hydrophobic residues downstream of phosphorylation site

23. Figure 5: ROP18 targets a conserved motif in SWI of IRGs • Explore possible sites for phosphorylation • SWI has the most high scoring motif among these IRG proteins ClustalX alignment of IRGs Sequence logo of region surrounding SWI

24. Figure 5: ROP18 targets a conserved motif in SWI of IRGs • ROP18 phosphorylates several Thr residues within a peptide derived from SWI of Irgb6 in vitro • Confirm the GAAPTp sequence was phosphorylated. • ROP18 phosphorylates IRGs in vitro at a conserved site within the SWI region

25. Is ROP18 necessary for macrophage survival?

26. Figure 6: ROP18 is necessary for macrophage survival Deletion of ROP18 Survival Rate Deletion of ROP18 resulted in increased recruitment of Irgb6 in IFNγ-activated macrophages

27. Figure 6: Irgb6 recruitment is necessary for maximal clearance of parasites Severe drop of clearance ability Confirm necessity of Irgb6 recruitment in maximal clearance of parasites

28. Summary • ROP18 phosphorylates a critical Thr residue on SWI, rendering IRGs incapable of oligomerization and blocking loading onto the parasite-containing vacuole.