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Role of AMA1 Cleavage in Toxoplasma Replication Switch

Intramembrane cleavage of AMA1 shifts Toxoplasma from invasive to replicative mode, implicating AMA1 in host recognition. AMA1 shedding aids in switching roles, investigated through ROM4 cleavage mutants. Experimental design includes AMA1 mutants and rescue studies with AMA1 tails. Conclusions highlight AMA1 cleavage as crucial for successful replication, with ROM4 mutants struggling in subsequent replicative cycles.

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Role of AMA1 Cleavage in Toxoplasma Replication Switch

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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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