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Santos, et al. Dec 23, 2010

Intramembrane Cleavage of AMA1 Triggers Toxoplasma to Switch from an Invasive to a Replicative Mode. Santos, et al. Dec 23, 2010. Invasion Steps in Toxoplasma. Each follows the general pattern of bind, orient, enter, replicate. Membrane Protein Shedding.

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Santos, et al. Dec 23, 2010

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  1. Intramembrane Cleavage of AMA1 Triggers Toxoplasma to Switch from an Invasive to a Replicative Mode Santos, et al. Dec 23, 2010

  2. Invasion Steps in Toxoplasma • Each follows the general pattern of bind, orient, enter, replicate

  3. Membrane Protein Shedding • After micronemeexocytosis, some membrane proteins, including AMA1, are cleaved by a group of intramembrane proteases known as rhomboids • Protein shedding thought to just be a result of the cell changing roles from invasive to replicative forms

  4. Replication and Division in Toxoplasma and Plasmodium

  5. Role of AMA1 • Believed to be involved in host cell recognition and binding • AMA1 KO’s produce cells incapable of secretion of rhoptries

  6. Critical Questions • Does AMA1 serve a secondary role as a moderator for switching Toxoplasma/Plasmodium from an invasive to a replicative state? • Is the intramembrane cleavage of AMA1 significant in this secondary role? • If so, at what point does a lack of AMA1 cleavage arrest during replication?

  7. Experimental Design • Create a mutant where AMA1 is unable to be cleaved by ROM4 • Overexpress the tail of AMA1 and see if it can restore normal replicative function

  8. FKBP and Shld-1 • FKBP binds a rapamycin analogue • Shld-1 is the nontoxic analogue of rapamycin • Within FKBP is a domain that, unless bound to Shld-1, will destabilize and target the protein for degradation • By fusing a dominant negative ROM4 to FKBP and overexpressing… • Endogenous ROM4 becomes ineffective upon Shld1 addition • AMA1 cannot be cleaved

  9. FKBP and Shld-1 FKBP domain Shld-1 ROM4 domain Ser to Ala mutation WT ROM4 AMA1 PM Cleaved AMA1 tails

  10. Expression of ROM Constructs

  11. Replication in ROM Constructs • ddROM4s-a expressing cells fail to form plaques

  12. Replication in ROM Constructs • ddROM4s-a mutant expressing cells arrest after 1-2 rounds of replication

  13. ddROM4s-a Replication Arrest • ddROM4s-a mutant expressing cells display signs of arrest during replication

  14. ddROM4s-a Defects Post-Replication N-Nucleus R-Rhoptries DG-Dense Granules C-Conoid Mi-Mitochondria MN-Micronemes RB-Residual Body PP-Posterior Pole

  15. Effects of Defective ROM4 Before and After Invasion • ddROM4s-a acting as a dominant negative is reversible (A) and invasion independent (B)

  16. Can ROM4 Defects be Rescued? • A series of AMA1 tails was made using the FKBP/Shld1 system. • Some tails were mutants, one was WT, all shared a sequence thought to be important for function (PSDLMQEAEPS)

  17. Conditional AMA1 Mutant Analysis

  18. Rescue with AMA1 Tail Addition

  19. Conclusions • AMA1 cleavage by ROM4 is required for successful replication in Toxoplasma/Plasmodium • The cleaved portion of AMA1 serves a role in initiating a fully fledged replicating parasite • ROM4 mutant cells struggle to undergo more than one replicative cycle, and exhibit signs of arrest and breakdown of various organelles

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