1. THE YERSINIA YSC-YOP ‘TYPE III’ WEAPONRY G.R. Cornelis, 2002. Nature Reviews in Molecular Cell Biology 3: 742-752.

2. TYPE III SECRETION What is it? What is it? Why is it important? Why is it important? Who does it? Who does it?

3. TYPE III SECRETION Bacteria inject proteins across cellular membranes into the host cell cytosol that have complex effects on intracellular systems. Bacteria inject proteins across cellular membranes into the host cell cytosol that have complex effects on intracellular systems. Type III secretion systems (TTSS) are found in a variety of animals, plants, and insects but the evolution is unclear. Type III secretion systems (TTSS) are found in a variety of animals, plants, and insects but the evolution is unclear. TTSS are often plasmid-encoded. TTSS are often plasmid-encoded.

4. Yersinia TTSS Yops – Yersinia outer proteins Yops – Yersinia outer proteins Ysc – Yop secretion apparatus Ysc – Yop secretion apparatus Effectors – Yops that enter the host cell and alter host cell functioning. Effectors – Yops that enter the host cell and alter host cell functioning. Injectisome – bacterial organelle that secretes the effector proteins into the host cell. Injectisome – bacterial organelle that secretes the effector proteins into the host cell.

5. Yersinia TTSS Overview Yersinia adhere to cell surface and deliver Yops into host cell cytosol with injectisome. Yersinia adhere to cell surface and deliver Yops into host cell cytosol with injectisome. Yops functions: Yops functions: 1) cytoskeletal effects interfere with phagocytosis. 1) cytoskeletal effects interfere with phagocytosis. 2) inhibit production of cytokines, chemokines, and adhesion molecules. 2) inhibit production of cytokines, chemokines, and adhesion molecules. Yersinia are better able to survive and multiply in lymphoid tissues by using the TTSS. Yersinia are better able to survive and multiply in lymphoid tissues by using the TTSS.

6. Yops Translocator Yops: Translocator Yops: YopB, YopD, LcrV form the pore for delivery. YopB, YopD, LcrV form the pore for delivery. Effector Yops: Effector Yops: YopH, YopE, YopT, YpkA/YopO – cytoskeletal disturbances, inhibition of phagocytosis. YopH, YopE, YopT, YpkA/YopO – cytoskeletal disturbances, inhibition of phagocytosis. YopP – downregulation of inflammatory response. YopP – downregulation of inflammatory response. YopM – function uncertain. YopM – function uncertain.

7. INJECTISOME Basal body with protein pump (YscN) and a central pore (YscC). Basal body with protein pump (YscN) and a central pore (YscC). Needle-like projection (YscF) for Yops delivery. Needle-like projection (YscF) for Yops delivery. From: Cornelis, G.R., 2002. Yersinia Type III secretion: send in the effectors. Journal of Cell Biology 158 (3): 401-408.

8. INJECTISOME Translocator YopB, YopD, LcrV are required to form pores for cell delivery. Translocator YopB, YopD, LcrV are required to form pores for cell delivery. Some Yops require chaperone proteins. Some Yops require chaperone proteins. YopN, TyeA, LcrG plug the injectisome channel before Yops delivery to cell. YopN, TyeA, LcrG plug the injectisome channel before Yops delivery to cell. Cell adhesion required for injectisome function. Cell adhesion required for injectisome function.

9. INJECTISOME Does the injectisome ‘needle’ pierce the host cell membrane, retract, or break down? Does the injectisome ‘needle’ pierce the host cell membrane, retract, or break down? Maybe translocator Yops destabilize host cell membranes after adhesion to allow piercing? Maybe translocator Yops destabilize host cell membranes after adhesion to allow piercing? Once the pore is formed, even non-effector proteins can be delivered to the host cell! Once the pore is formed, even non-effector proteins can be delivered to the host cell!

10. From: Cornelis, G.R., 2002. Yersinia Type III secretion: send in the effectors. Journal of Cell Biology 158 (3): 401-408.

11. YOPS EFFECTS Phagocyte paralysis Phagocyte paralysis Reduction in pro-inflammatory response Reduction in pro-inflammatory response Inhibition of lymphocyte proliferation Inhibition of lymphocyte proliferation

12. PHAGOCYTE PARALYSIS Synergistic effects of Yops H, E, T, and O. Synergistic effects of Yops H, E, T, and O. YopH – a phosphotyrosine phosphotase (PTPase) YopH – a phosphotyrosine phosphotase (PTPase) Dephosphorylates focal adhesion protein p130. Dephosphorylates focal adhesion protein p130. Dephosphoylates scaffolding proteins SKAP-HOM. Dephosphoylates scaffolding proteins SKAP-HOM. Suppresses oxidative burst in macrophages. Suppresses oxidative burst in macrophages. Reduces calcium signals needed for phagocytosis. Reduces calcium signals needed for phagocytosis.

13. PHAGOCYTE PARALYSIS YopE – a GTPase activating protein (GAP) that hydrolyzes Rho, Rac, Cdc42 to ‘off’ position. YopE – a GTPase activating protein (GAP) that hydrolyzes Rho, Rac, Cdc42 to ‘off’ position. YopT – a cysteine protease that inactivates Rho, Rac, Cdc42 by cleaving from plasma membrane. YopT – a cysteine protease that inactivates Rho, Rac, Cdc42 by cleaving from plasma membrane. YpkA/YopO – an autophosphorylating serine/threonine kinase that inhibits Rho and Rac by an unknown mechanism. YpkA/YopO – an autophosphorylating serine/threonine kinase that inhibits Rho and Rac by an unknown mechanism.

14. From: Cornelis, G.R., 2002. Yersinia Type III secretion: send in the effectors. Journal of Cell Biology 158 (3): 401-408.

15. INHIBITING INFLAMMATION YopP – possibly a SUMO protease. YopP – possibly a SUMO protease. Inhibits the IKKB that would phosphorylate IKB to release inhibition of NF-KB. Because NF-KB is inhibited, PMN recruitment decreases due to: Inhibits the IKKB that would phosphorylate IKB to release inhibition of NF-KB. Because NF-KB is inhibited, PMN recruitment decreases due to: Decreased TNF release by macrophages. Decreased TNF release by macrophages. Decreased IL-8 release from epithelial and endothelial cells. Decreased IL-8 release from epithelial and endothelial cells. Decreased ICAM and E-selectin adhesion molecule expression on endothelial cells. Decreased ICAM and E-selectin adhesion molecule expression on endothelial cells.

16. INHIBITING INFLAMMATION Other YopP effects: Other YopP effects: Inhibition of MAPK kinases that keep the CREB transcription factor from being activated. Inhibition of MAPK kinases that keep the CREB transcription factor from being activated. Induction of macrophage apoptosis by cleavage of the Bcl-2 protein Bid, release of cytochrome c from mitochondria, and activation of caspases. Induction of macrophage apoptosis by cleavage of the Bcl-2 protein Bid, release of cytochrome c from mitochondria, and activation of caspases.

17. INHIBITING INFLAMMATION YopH – in addition to antiphagocytic activities: YopH – in addition to antiphagocytic activities: Inhibition of the PI3K/Akt pathway, causing: Inhibition of the PI3K/Akt pathway, causing: Reduced MCP1 synthesis that is needed to recruit macrophages to lymph nodes. Reduced MCP1 synthesis that is needed to recruit macrophages to lymph nodes. Reduced T cell ability for cytokine production. Reduced T cell ability for cytokine production. Reduced B cell ability to upregulate surface costimulatory molecules such as CD86. Reduced B cell ability to upregulate surface costimulatory molecules such as CD86.

18. From: Cornelis, G.R., 2002. Yersinia Type III secretion: send in the effectors. Journal of Cell Biology 158 (3): 401-408.

19. MYSTERIOUS YOPS YopM – important virulence factor in mice. YopM – important virulence factor in mice. Subunits form a hollow cylinder. Subunits form a hollow cylinder. Uses a vesicle associated pathway to migrate to cell nucleus and inhibit transcription of genes involved in cell growth and proliferation. Uses a vesicle associated pathway to migrate to cell nucleus and inhibit transcription of genes involved in cell growth and proliferation. LcrV – in addition to translocation of Yops: LcrV – in addition to translocation of Yops: Promotes IL-10 production that suppresses TNF production in macrophages. Promotes IL-10 production that suppresses TNF production in macrophages. IL-10 deficient mice are more resistant to Yersinia infection but mechanisms are difficult to study. IL-10 deficient mice are more resistant to Yersinia infection but mechanisms are difficult to study.

20. From: Cornelis, G.R., 2002. Yersinia Type III secretion: send in the effectors. Journal of Cell Biology 158 (3): 401-408.

21. CONCLUSIONS Translocator Yops – B, D, LcrV. Translocator Yops – B, D, LcrV. Effector Yops – Yop H, E, T, O, P, M. Effector Yops – Yop H, E, T, O, P, M. Yersinia Ysc-Yop TTSS has complex effects: Yersinia Ysc-Yop TTSS has complex effects: Inhibition of phagocytosis. Inhibition of phagocytosis. Reduced recruitment of PMNs and macrophages. Reduced recruitment of PMNs and macrophages. Inhibition of lymphocyte proliferation. Inhibition of lymphocyte proliferation.