PfERC knockdown inhibits PVM breakdown. In these parasite lines, the endogenous locus of PfERC was tagged with the inducible version of the ribozyme gene, glmS, or with the inactive version of the ribozyme, M9 (termed PfERC-glmS and PfERC-M9, respectively). (C) Representative SEM images of C2-arrested PfERC-glmS (n=4 biological replicates) and PfERC-M9 (n=4 biological replicates) mutants fixed immediately after washing off C2 and after 30 min as described for panel A. During this time period, PfERC-M9 schizonts were able to initiate egress after removal of C2. We observed free PfERC-M9 merozoites attached to the RBCs as well as clusters of merozoites that had broken out of the PVM but appeared to be contained by a collapsed RBCM wrapped around them. In contrast, the PfERC-glmS schizonts were still stuck within the RBC and looked identical to the C2-arrested schizonts, suggesting that they had not initiated egress even though the PKG activity was no longer inhibited (C). These data suggest that knockdown of PfERC blocks egress at an early step, perhaps blocking the rupture of the PVM. (D) Representative TEM images of PfERC-glmS (n=2 biological replicates) and PfERC-M9 (n=2 biological replicates) schizonts incubated with compound 1 as described for panel A. Bar, 800 nm. PVM is marked by black arrowheads. PfERC-glmS and PfERC-M9 schizonts were morphologically identical and, as expected, had formed merozoites within the PVM and RBCM (D). C1 was washed off these schizonts and the schizonts were observed by the use of TEM after 30 min (D). Results show that the PfERC-M9 schizonts were able to break down the PVM within 30 min.

Fierro MA, Asady B, Brooks CF, Cobb DW, Villegas A, Moreno SNJ, Muralidharan V. An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites. mBio. 2020 11(1). pii: e03078-19.