INVESTIGADORES
PARUSSINI GIMENEZ silvana fabiola
congresos y reuniones científicas
Título:
Functional role of the intramembrane proteolytic cleavage of Toxoplasma gondii AMA1 mediated by rhomboid proteases
Autor/es:
FABIOLA PARUSSINI; JEFFREY MITAL; SYED M. MOIN; SINISA URBAN; GARY E. WARD
Lugar:
Kerkrade, Netherland
Reunión:
Congreso; 10th International Congress of Toxoplasmosis; 2009
Resumen:
Apical membrane antigen 1 (AMA1) is a conserved type 1 transmembrane protein that localizes to the micronemes of apicomplexan parasites, is secreted onto the parasite plasma membrane and shed during invasion. Phenotypic characterization of TgAMA1 conditional knockout parasites has demonstrated that TgAMA1 plays a critical role during T. gondii invasion as a secondary adhesin and/or rhoptry secretion trigger (Mital et al., Mol Biol Cell 16: 4341). To identify the requirements for cleavage of the TgAMA1 transmembrane domain (TMD; NTALIAGLAVGGVLLLALLGGGC) by the rhomboid protease TgROM5, we have introduced mutations in both helix-breaking motifs AG and GG, or replaced the TMD with the TMD from an unrelated protein, TGFa. The four cleavage mutants (CMs) generated were:  CM1, AG>FF; CM2, GG>FF; CM3, AG>FF and GG>FF; and CM4, NTALIAGLAVGG>ITALVVVSIVAL. COS cells were cotransfected with TgROM5 and either wild type (WT) or mutant TgAMA1, and the supernatant from the cells was examined by western blotting for the release of the TgAMA1 ectodomain. CM2 had no detectable effect on cleavage, CM1 showed significantly reduced cleavage, and CM3 and CM4 blocked cleavage completely. To elucidate the function of TgAMA1 cleavage and ectodomain shedding during invasion, we have generated epitope-tagged TgAMA1 constructs containing either the WT TMD or the TMD mutations described above. These constructs were used to transfect TgAMA1 conditional knockout parasites. While stably transfected clones expressing CM1 and CM2 were readily obtained, we failed after repeated attempts to isolate viable parasites stably expressing CM3 or CM4. These results suggest that mutations that completely block the TgROM5-mediated cleavage of TgAMA1 cannot be tolerated by the parasites. We are currently attempting to characterize the invasion phenotype of parasites transiently expressing the CM3 and CM4 mutations.