Apical membrane antigen 1 (AMA1) is normally a conserved transmembrane adhesin of apicomplexan parasites that plays an important role in host-cell invasion. expressing these TgAMA1 variants. Contrary to expectation, variants that improved or decreased TgAMA1 processing by >10-collapse experienced no phenotypic effects, exposing the levels of rhomboid proteolysis in parasites are not delicately balanced. Only parasites transgenically expressing or transporting 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 1418033-25-6 supplier demonstrate that TgAMA1 cleavage plays a role in invasion, but refute a recently proposed 1418033-25-6 supplier model in which parasite replication within the sponsor cell is regulated by intramembrane proteolysis of TgAMA1. AMA1 (TgAMA1) and several other microneme proteins is mediated from the rhomboid class of intramembrane proteases: rhomboid-mediated cleavage of these proteins has been proven in cell-based reconstitution assays (16), and the cleavage sites within the transmembrane website (TMD) of TgAMA1 (17), TgMIC2 (18), and TgMIC16 (19) have been mapped in vivo. Rhomboid proteases will also be capable of cleaving over a dozen adhesins implicated in malaria parasite invasion (20). Of the rhomboid proteases, TgROM4 and TgROM5 localize to the parasite plasma membrane and are likely to be responsible for microneme protein cleavage and dropping (19, 21, 22). TgROM5 is definitely active against the TMDs of several microneme proteins in heterologous cleavage assays (21), and TgROM4, although inactive in such assays (21), cleaves particular microneme proteins in vivo (23). Why TgAMA1 is definitely cleaved and shed from the surface of invading parasites remains a major unresolved query. Intramembrane cleavage of microneme proteins may be important for disengaging relationships between parasite surface adhesins and host-cell ligands during invasion (24), but this model offers proven difficult to test. Depletion of TgROM4 in conditional knockout (TgROM4KOi) parasites results in the defective processing of multiple microneme proteins and does indeed impair host-cell invasion, but the crucial substrate(s) for invasion have not been recognized (23). Although no effect on parasite replication was observed in the TgROM4KOi parasites, transgenic parasites expressing a catalytically inactive TgROM4 (ddTgROM4S-A) showed both a moderate effect on invasion and seriously impaired intracellular replication (25). Insufficient TgAMA1 cleavage and launch of its C-terminal tail into the parasite cytosol were proposed as the cause of the replication defect because cytosolic overexpression of the TgAMA1 C-tail rescued the phenotype (25). It was assumed the catalytically inactive ddTgROM4S-A protein sequestered TgAMA1 from your endogenous active enzyme (25), although ddTgROM4S-A protein levels were comparable to active TgROM4 and neither substrate trapping nor any defect in the control or dropping of TgAMA1 was shown. In the current study, we wanted to determine directly the part that intramembrane proteolysis of TgAMA1 takes on in the parasites asexual existence cycle. We used a quantitative heterologous cleavage assay to define motifs within the TgAMA1 TMD that are important for rhomboid-mediated control and then indicated cleavage mutants either inside a TgAMA1 conditional knockout parasite collection or by knock-in in the locus. Phenotypic analyses of these parasites exposed that cleavage of TgAMA1 is required for efficient host-cell invasion, but argue strongly against a role Rabbit Polyclonal to DIL-2 for TgAMA1 cleavage in controlling intracellular replication. Results and Conversation Development of a System for Studying TgAMA1 Intramembrane Cleavage. To study the function of TgAMA1 cleavage, we used TgAMA1 conditional knockout parasites (TgAMA1KOi) in which the manifestation of TgAMA1, Myc-tagged at its C terminus, can be experimentally down-regulated by treatment with anhydrotetracycline (ATc) (5). With this background, we created a new parasite collection (designated AMA1WT) stably expressing a second copy of under the control of its endogenous promoter and having a Flag tag inserted into website 2 of its ectodomain (Fig. S1). As expected, the manifestation of TgAMA1-Myc was down-regulated in response to ATc in both the TgAMA1KOi and AMA1WT parasites (Fig. 1and transgene was insensitive to ATc (Fig. 1and and and Fig. S1), as previously mapped in tachyzoites (17). The heterologous cleavage assay consequently efficiently and faithfully reconstitutes TgAMA1 processing. Fig. 2. Recognition of TgAMA1 mutations that impact rhomboid proteolysis inside a heterologous cleavage assay and in parasites. (and Fig. S1) and used quantitative Western analysis to assess their importance for cleavage. TgAMA1 processing does not conform to the consensus recently identified for any subset of rhomboid proteases (26) because it contains an alanine four residues before the cleavage site, and mutating L461 two residues after the cleavage site to glycine dramatically cleavage by 13-fold (Fig. 2and and < 0.0001). These data reveal that almost half of the invasion defect observed in TgROM4-depleted parasites (23) could 1418033-25-6 supplier be due.