Rhomboid-mediated intramembrane proteolysis of Toxoplasma gondii AMA1 (TgAMA1) facilitates host cell invasion but is dispensable for intracellular replication.

FABIOLA PARUSSINI; QING TANG; SYED M. MOIN; SINISA URBAN; GARY E. WARD

Ottawa

Congreso; 11th International Congress on Toxoplasmosis; 2011

Apical membrane antigen 1 (AMA1) is aconserved and essential transmembrane adhesin of apicomplexan parasites thatfunctions in organization of the moving junction between the parasite and hostcell during invasion. Like other microneme-derived adhesins, AMA1 isproteolytically processed and shed from the surface of invading parasites. Toelucidate the function of T gondii AMA1(TgAMA1) intramembrane cleavage and shedding, we used a heterologous cleavageassay to identify mutations within the TgAMA1 transmembrane domain (LIAGLAVGGVLLLALLGGGCYFA)that inhibit cleavage by the cell surface rhomboid protease TgROM5.Quantitative analysis revealed that the TgAMA1AG/FF mutation reducedcleavage by 20-fold, while the TgAMA1GG/FF mutation reduced cleavageby less than 2-fold. Mutating both of these motifs in a quadruple mutantreduced cleavage to undetectable levels (at least 215-fold), as did replacingthe first half of the TgAMA1 TMD with that from the human growth factor, TGFa. We then generatedTgAMA1 expression constructs containing these mutations and used them tocomplement a TgAMA1 conditional knockout parasite line. Parasites stablyexpressing the noncleavable TgAMA1AG/FF +GG/FF mutation wereoutcompeted in growth assays by parasites expressing an equivalent amount ofwild-type TgAMA1.  We subsequently determinedthat the TgAMA1 cleavage mutation had no effect on intracellular replicationbut inhibited invasion by 30%. These data demonstrate that AMA1 cleavage playsa role in invasion, and they argue against a recently proposed model in which successiverounds of parasite replication within the host cell are regulated by intramembranecleavage of TgAMA1.