Mutant/mutation
The mutant expresses Py-ICP fused to a Regulated Fluorescent Affinity (RFA) tag containing a GFP cassette and HA epitope tag  (Muralidharan et al., 2011).

In addition to a GFP cassette, the RFA tag contained an HA epitope and an E. coli DHFR degradation domain, which destabilizes the protein in the absence of folate analogs, including trimethoprim (TMP) (Muralidharan et al., 2011).

ICP is conserved among numerous Plasmodium species. However, a Py-ICP ortholog had not been annotated in PlasmoDB (www.plasmodb.org). To determine if Py contained an ICP ortholog, a BLAST search of the Py genome was conducted using Pf-ICP as the query and observed highly conserved nucleotide sequences at the C-terminal region of a 7.3 kb gene, PY03424. It was found that that PY03424 was composed of two separate genes: a single exon gene is termed PY03424* and Py-ICP. The re-annotated Py-ICP gene has 85% and 34% amino acid identity to its orthologs in Pb and Pf, respectively.

Protein (function)
An endogenous cysteine protease inhibitor has been identified in P. falciparum, Pf-ICP (PF3D7_0911900; Falstatin), via a BLAST search of the Pf genome using the Trypanosoma cruzi cysteine protease inhibitor chagasin as a query. A recombinant  Pf-ICP expressed in E. coli was shown to potently inhibit a number of host proteases by in vitro protease activity assays. Additionally, Pf-ICP also inhibited several parasite proteases in these assays, including falcipain-2 and falcipain-3, but not falcipain-1.

Phenotype
Py-ICP-RFA expressed in blood stages

Additional information
To determine if Py-ICP-RFA could be destabilized in vivo, we monitored the average fluorescence intensity of GFP from RFA tagged parasites over time.

TMP was administered in the drinking water of mice 24 hrs prior to inoculation with Py-ICP-RFA infected erythrocytes. Two days post infection drug was removed from a subset of mice, while the remaining mice were maintained on TMP for four additional days. Py-ICP-RFA was propagated in SW mice whose water contained 250µg/ml of TMP (Sigma).

The average fluorescence intensity of GFP from 5x107 Py-ICP-RFA infected erythrocytes was determined at varying time points using a fluorescence microplate reader. After removal of TMP, GFP fluorescence intensity from Py-ICP-RFA parasites decreased over time, reaching a maximum reduction of 30% as compared to TMP stabilized protein by 72 and 96 hr. Protein degradation was also monitored by western blot, which revealed up to a 50% decrease in total ICP protein levels during blood stage replication in vivo. Both the full length and processed form of Py-ICP were reduced following removal of TMP. However, this down-regulation was more pronounced in the processed form, which exhibited more than a 5-fold decrease as compared to a 1.7-fold reduction of full length Py-ICP.

However, this reduction was not sufficient to decrease the viability of the recombinant parasite, as there was no detectable difference in blood stage growth in vivo in the absence of TMP.

Thus, a larger decrease in protein expression is likely necessary to impact parasite replication and reveal a phenotype of ICP depletion in blood stage parasites.

Other mutants
See RMgm-970 for successful disruption of the icp gene!!!
See also RMgm-245, RMgm-397 and RMgm-844 for unsuccessful attempts to disrupt the P. berghei icp gene

See RMgm-845 for a mutant expressing a normal ICP and a quadruple C-myc tagged version of ICP.