Mutant/mutation
The mutant lacks expression of Scd and expresses GFP (under a constitutive promoter).

Protein (function)
The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Plasmodium parasites can modify de novo synthesized or scavenged fatty acids into long unsaturated fatty acids. An endoplasmic reticulum (ER)-localized stearoyl-CoA desaturase (Scd) was identified in P. falciparum that catalyzes the formation of oleic acid from stearic acid by the insertion of a cis double bond at the Δ9 position of fatty acyl-CoAs.

Phenotype
Scd KO sporozoites infect the liver but fail to establish blood-stage infections in mice.
Scd KO sporozoites were allowed to invade HepG2 cells and sporozoites present inside and outside of the cells were counted, which revealed normal invasion by KO sporozoites. To analyze EEF development progress in vivo, infected mouse livers were harvested at 36, 55, and 72 hpi, and parasite burden was quantified by measuring 18S rRNA copy number using real-time PCR. We found that the 18S rRNA copy number was comparable in WT GFP and KO parasites at 36 hpi, but it was significantly lower at 55 hpi in KO parasites than in WT GFP parasites. However, the 18S rRNA copy number at 72 hpi was significantly lower in WT GFP than in KO, which suggested that mature WT GFP parasites egress from the liver but that KO parasites failed to mature and remained in the liver. We confirmed the maturation defect by determining the merozoite surface protein 1 (MSP1) transcripts at 55 hpi and found that it was significantly decreased in the KO parasites.

Scd KO liver stages grow normally but do not form hepatic merozoites.
HepG2 cells were infected with sporozoites, and the culture was fixed at different time points and stained with anti-UIS4 antibody and Hoechst. The Scd KO parasites grew similarly to the WT GFP parasites during liver-stage development. We counted the EEF numbers and measured the size at 36, 48, and 62 hpi and found comparable numbers and sizes in Scd KO and WT GFP parasites. Next, we checked the formation of merozoites by staining with an anti-MSP1 antibody. WT GFP showed nuclear segregation and merozoite formation. In contrast, there was abnormal DNA segregation in Scd KO parasites and merozoite formation was abolished. We counted the nuclei in the EEF, which were found to be significantly fewer in Scd KO parasites.

Additional information
Analysis of a mutant expressing a C-terminal 3xHA tagged version of Scd (RMgm-5551) showed the following: Scd was expressed in the blood stage and post 48 h in the liver stage. P. berghei Scd primarily colocalized to the ER in asexual blood stages and partially during liver stages.

To evaluate organelle morphology in Scd KO parasites, EEFs fixed at 62 hpi were immunostained with anti-ACP or anti-Bip antibodies to visualize apicoplast and endoplasmic reticulum branching, respectively. We found normal branching of the apicoplast and endoplasmic reticulum in WT GFP parasites, whereas it was impaired in Scd KO parasites

Other mutants