期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2012
卷号：109
期号：19
页码：7463-7468
DOI：10.1073/pnas.1114661109
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Apical membrane antigen 1 (AMA1) is a conserved transmembrane adhesin of apicomplexan parasites that plays an important role in host-cell invasion. Toxoplasma gondii AMA1 (TgAMA1) is secreted onto the parasite surface and subsequently released by proteolytic cleavage within its transmembrane domain. To elucidate the function of TgAMA1 intramembrane proteolysis, we used a heterologous cleavage assay to characterize the determinants within the TgAMA1 transmembrane domain (ALIAGLAVGGVLLLALLGGGCYFA) that govern its processing. Quantitative analysis revealed that the TgAMA1L/G mutation enhanced cleavage by 13-fold compared with wild type. In contrast, the TgAMA1AG/FF mutation reduced cleavage by 30-fold, whereas the TgAMA1GG/FF mutation had a minor effect on proteolysis; mutating both motifs in a quadruple mutant blocked cleavage completely. We then complemented a TgAMA1 conditional knockout parasite line with plasmids expressing these TgAMA1 variants. Contrary to expectation, variants that increased or decreased TgAMA1 processing by >10-fold had no phenotypic consequences, revealing that the levels of rhomboid proteolysis in parasites are not delicately balanced. Only parasites transgenically expressing or carrying a true knock-in allele of the uncleavable TgAMA1AG/FF+GG/FF mutant showed a growth defect, which resulted from inhibiting invasion without perturbing intracellular replication. These data demonstrate that TgAMA1 cleavage plays a role in invasion, but refute a recently proposed model in which parasite replication within the host cell is regulated by intramembrane proteolysis of TgAMA1.
