Mutant/mutation
In the mutant the P. berghei pimms43 gene has been replaced by the pimms43 gene of P. falciparum

Protein (function)
PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43).
PIMMS43 encode deduced proteins of 505 and 350 amino acids, respectively. N-terminal signal peptides (amino acids 1 to 25 for PfPIMMS43 and 1 to 22 for PbPIMMS43) and C-terminal transmembrane domains (amino acids 482 to 504 for PfPIMMS43 and 327 to 350 for PbPIMMS43) are predicted for both proteins. The transmembrane domains are predicted by PredGPI to also contain signals for attachment of a GPI lipid anchor with 99% probability.

Our transcriptomic profiling of field P. falciparum isolates from Burkina Faso in the midgut of sympatric Anopheles coluzzii (previously Anopheles gambiae M form) and Anopheles arabiensis mosquitoes and a laboratory P. berghei strain in the midgut of A. coluzzii identified hundreds of genes exhibiting conserved and differential expression during gametocyte-to-oocyst development. Several of them encoding putatively secreted or membraneassociated proteins were made part of a screen to identify genes that function during parasite infection of the mosquito midgut. These genes were given a candidate gene number preceded by the acronym PIMMS, for “Plasmodium Infection of the Mosquito Midgut Screen.”
Here, we report the characterization of P. falciparum and P. berghei PIMMS43 that encodes a membrane-bound protein found on the surface of ookinetes and sporozoites. The gene was first reported in P. berghei to be a target of the transcription factor AP2-O, has a role in mosquito midgut invasion and oocyst development, and was named POS8. A later study by another group reported the gene as being important for ookinete maturation, designating it as PSOP25. Evidence is presented that PIMMS43 has no detectable function in ookinete maturation or mosquito midgut invasion but plays a key role in ookinete evasion of the mosquito complement-like response.

Phenotype
Analysis of mutants lacking PIMMS43 (RMgm-4824, RMgm-4825) showed the following:
Normal blood stage development; normal production of gametocytes, gametes and ookinetes. In Anopheles coluzzi no oocyst formation; no sporozoite formation (in A stephensi low numbers of oocysts were observed, but no sporozoites).

We also raised a rabbit polyclonal antibody against a codonoptimized coding fragment of P. falciparum PIMMS43 (amino acids 25 to 481) expressed in E. coli cells and lacking the predicted signal peptide and C-terminal transmembrane domain (α-Pfc43opt). We examined the affinity and specificity of this antibody by generating and using a P. berghei c507 transgenic line (PbPfc43), where PbPIMMS43 was replaced by PfPIMMS43. PCR genotypic analysis confirmed successful modification of the endogenous PbPIMMS43 genomic locus, and RT-PCR analysis confirmed that PfPIMMS43 is transcribed in in vitro-cultured P. berghei ookinetes. Western blot analysis of total protein extracts prepared from purified in vitro-cultured PbPfc43 ookinetes using the α-Pfc43opt antibody revealed a strong band of ∼60 kDa, corresponding to the predicted molecular weight of the deduced PfPIMMS43 protein. PfPIMMS43 prominently localizes on the surface of female gametes or early stage zygotes found in the A. coluzzii blood bolus , as well as on the surface of ookinetes traversing the mosquito midgut epithelia and sporozoites found in the mosquito salivary gland lumen.

Next, we investigated whether PfPIMMS43 could complement the function of its P. berghei ortholog by infecting naïve A. coluzzii mosquitoes with the PbPfc43 parasite line and counting the number of oocysts detected in the mosquito midguts. Infections with c507 and Δc43 parasites served as positive and negative controls, respectively. The results showed that the PbPfc43 line exhibited an intermediate phenotype compared to c507 and Δc43 both in terms of both infection prevalence and intensity. Oocysts were morphologically variable and smaller in size compared to c507 oocysts and contained a small number of sporozoites that could not be transmitted to a new mouse host, resembling the phenotype obtained with Δc43 infections following silencing of the mosquito complement-like system. We examined whether this partial complementation phenotype could be affected upon LRIM1 silencing. Indeed, a significant increase in both the infection prevalence and oocyst numbers was observed , yet oocysts remained small and morphologically variable and produced few sporozoites. These results suggest that PfPIMMS43 can only partly complement the function of its PbPIMMS43 ortholog and corroborate the dual function of PIMMS43 in ookinete to oocyst transition and in oocyst maturation and sporozoite development, respectively.

Additional information
Evidence is presented that:
- Ookinetes Lacking PIMMS43 Are Killed by the Mosquito Complement-Like Response upon Midgut Traversal.
- PIMMS43 Is Additionally Required for Oocyst Maturation and Sporozoite Development
- PIMMS43 KO Leads to Compromised Ookinete Fitness and Attack by the Complement-Like Response

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