Mutant/mutation
The mutant c22::gfp lacks expresses a C-terminal GFP-tagged version of PIMMS22

Protein (function)
PfPIMMS22 encodes a 393 amino acid (45 kDa) protein and PbPIMMS22 encodes a 393 aa (44 kDa) protein, both with no predicted signal peptide or transmembrane domain. The protein is highly conserved amongst Plasmodium orthologues. PyPIMMS22 (PY17X_1424900) was previously identified in salivary gland sporozoites through a  subtractive hybridization (SSH) profiling and termed sporozoite protein S15. The same protein was also identified in midgut oocyst sporozoites as an interacting partner to the apicomplexan specific RNA-binding protein, ALBA4, which is involved in mRNA regulation in gametocyte and midgut oocyst sporozoite development. PIMMS22 homologues are found in other apicomplexan parasites including Toxoplasma gondii, Neospora caninum and Eimeria with sequence identities to PIMMS22 ranging from 36% to 42%. InterPro domain analysis revealed no recognizable domain in PIMMS22.

Phenotype
See below for details of GFP-expression
See RMgm-4999 for a mutant lacking expression of PIMMS22, showing reduced oocyst and sporozoite formation

Additional information
Three genes were selected for analysis that exhibit highly abundant transcripts 24 h post blood feeding in the midguts of Anopheles coluzzii mosquitoes.
 
PIMMS01: PF3D7_0112100; PBANKA_0201700 
PIMMS57: PF3D7_1244500; PBANKA_1457700
PIMMS22: PF3D7_0814600; PBANKA_1422900 
((PIMMS: Plasmodium Infection of the Mosquito Midgut Screen)

PbPIMMS01 and PbPIMMS57 transcripts were highly abundant in purified mature ookinetes and not detectable in mixed blood stages and purified gametocytes. While PbPIMMS57 appears to be specific for ookinetes, low levels of PbPIMMS01 transcripts are also detected in mature oocysts and midgut sporozoites at 10 days and 15 days post blood-feeding. Like PfPIMMS22, expression of PbPIMMS22 starts in gametocytes and continues at high levels at 24 hour and peaks in midgut sporozoites at day 15.

Western blot analysis using the c22::gfp transgenic parasite line and a-GFP antibody revealed high levels of the PbPIMMS22::GFP fusion protein in ookinete and oocyst derived sporozoites, at the expected molecular weight of 71 kDa. This band was not detected in the ANKA 2.34 background parasite line. IFAs on the c22::gfp parasites allowed us to analyze the sub-cellular localization of the fusion protein. In Triton X-100 permeabilized blood stage gametocytes, PbPIMMS22::GFP was found to be localized on the cell periphery with the majority of the protein localizing in the cytoplasm. Non-Triton X-100 permeabilized blood stage gametocytes are visibly less fluorescent than their Triton X-100 permeabilized counterparts. In Triton X100 permeabilized in vitro ookinetes, PbPIMMS22::GFP was clearly observed on the ookinete periphery, a result that is inconsistent with the predicted absence of a signal peptide or transmembrane domain. Additional staining on non-Triton X-100 permeabilized ookinetes show some peripheral signal albeit much weaker than its Triton X-100 permeabilized counterpart suggesting that PbPIMMS22::GFP is mainly localized on the inner surface of the ookinete and not on the plasma membrane. IFAs on in vivo midgut epithelium invading ookinetes at 26 hpbf also show surface localization of PbPIMMS22::GFP. In midgut sporozoites, PbPIMMS22::GFP is found on the surface of both Triton X-100 and non-Triton X-100 permeabilized sporozoites.

From the abstract:
PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. 

Other mutants