INVESTIGADORES
PARUSSINI GIMENEZ silvana fabiola
congresos y reuniones científicas
Título:
Intramembrane proteolysis of Toxoplasma apical membrane antigen 1 is dispensable for invasion but plays an essential role in intracellular replication
Autor/es:
FABIOLA PARUSSINI; JEFFREY MITAL; SYED M. MOIN; SINISA URBAN; GARY E. WARD
Lugar:
Newport, Rhode Island
Reunión:
Conferencia; Gordon Research Conference: Biology of Host-Parasite Interactions; 2010
Institución organizadora:
Gordon Research Conferences
Resumen:
Several microneme proteins of T. gondii required for attachment and penetration are proteolytically processed on the parasite surface and shed during invasion. It has been postulated that the intramembrane cleavage of these proteins is mediated by one of the recently identified T. gondii rhomboid proteases and is required to disrupt molecular interactions between the parasite and host cell surface. Apical membrane antigen 1 (AMA1) is a conserved type 1 transmembrane protein that localizes to the micronemes of apicomplexan parasites and is secreted onto the parasite plasma membrane during invasion. Phenotypic characterization of TgAMA1 conditional knockout parasites has demonstrated that TgAMA1 is involved in the regulation of rhoptry secretion and in organizing the moving junction, a ring-shaped zone of tight contact between the invading parasite and the host cell membrane. We sought to analyze the role of TgAMA1 processing. Using a heterologous expression assay, we identified two motifs within the TgAMA1 transmembrane domain (TMD; NTALIAGLAVGGVLLLALLGGGC) that are required for cleavage by the rhomboid protease TgROM5.  Quantitative analysis revealed that the TgAMA1AG/FF mutant reduced cleavage by 20-fold, while the TgAMA1GG/FF mutant reduced cleavage by no more than 2-fold. Mutating both of these motifs together in a quadruple mutant reduced cleavage at least 215-fold to undetectable levels, as did replacing the top of the TgAMA1 TMD with that from the human growth factor, TGFa. To elucidate the function of TgAMA1 cleavage and ectodomain shedding during invasion, we generated TgAMA1 expression constructs corresponding to the mutants above to complement TgAMA1 conditional knockout parasites. While stably transformed clones expressing TgAMA1AG/FF and TgAMA1GG/FF were readily obtained, we failed after repeated attempts to isolate viable parasites stably expressing the uncleavable TgAMA1AG/FF+GG/FF or TgAMA1TGFa mutants. These results suggest that only mutations that completely block the TgROM5-mediated cleavage of TgAMA1 cannot be tolerated by the parasites. Unexpectedly, such mutations had no effect on host cell invasion, disrupting instead the progression of the parasites through mitosis. These data argue against the widely accepted model that microneme protein shedding is required solely for host cell invasion; rather, they reveal a new role for the intramembrane cleavage of TgAMA1 in intracellular parasite replication.