Mutant/mutation
The mutant expresses a c-terminally GFP-tagged version of PbICP (P. berghei inhibitor of cysteine proteases) under control of the eef1a promoter. This transgene was integrated by single cross-over into the c/d-ssu ribosomal locus.

Protein (function)
P. berghei express a cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). In the paper evidence is presented that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death (apoptosis). PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Biochemical analysis indicates that both full-length PbICP and the truncated C-terminal domain are inhibitors of cathepsin L-like host and parasite cysteine proteases. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. Falstatin/PfICP has been characterized as an inhibitor of various parasite and host cell cysteine proteases and is expressed by blood schizonts, merozoites and rings but not in trophozoites. PbICP is also expressed in blood stages.

In the paper it is reported that several attempts have been made to knock out the pbicp gene without success, suggesting that PBICP is essential for blood stage development (see RMgm-397).

Phenotype
Blood stages and liver stages express PbICP-GFP under control of the constitutive eef1a promoter. In the paper evidence is presented that the native PbCIP protein is posttranslationally processed. In blood stages the anti-PbICP-C antiserum detected the full-length protein (55 kDa) as well as the processed form after cleavage of the N-terminal extension domain (23 kDa), whereas anti-PbICP-N antiserum detected only the full-length protein of 55 kDa. To analyze processing of PbICP during liver stage development by western blotting, the mutant was generated that expresses a PbICP-GFP fusion protein under the control of the strong constitutive eef1a promotor. PbICP overexpression was necessary because infection rates of HepG2 cells are in general very low (2–10%) and do not allow detection of endogenous proteins by western blotting. Using this PbICP-overexpressing mutant for HepG2 cell infection, it was then possible to demonstrate PbICP processing before and after the detachment of infected hepatocytes at the end of the liver stage.

Additional information
Overexpression of the PbICP-GFP fusion protein had no negative effects on parasite development suggesting that the presence of the fusion protein is not toxic for exoerythrocytic parasites. Interestingly, transgenic parasites constitutively expressing PbICP-GFP showed a slightly but significantly increased level of HepG2 cell invasion compared to wild type parasites

Other mutants
RMgm-397: Unsuccessful attempts to disrupt the pbicp gene