Mutant/mutation
The mutant expresses a C-terminal eGFP-tagged version of SpdS

Protein (function)
Positively-charged polyamines are essential molecules for the replication of eukaryotic cells. Polyamines are essential, positively charged, ubiquitous aliphatic molecules with two or more amino groups, which include Putrescine, Spermidine and Spermine. Polyamines are essential, positively charged, ubiquitous aliphatic molecules with two or more amino groups, which include Putrescine, Spermidine and Spermine. Known parasitic protozoa that have the complete central polyamine pathway (AdoMetDC, ODC, and SpdS) are Plasmodium spp., Leishmania spp., and Trypanosoma brucei. However, in these species, due to the absence of Spermine Synthase, Spermidine Synthase most probably plays a promiscuous role and is responsible for the synthesis of both Spermidine and Spermine.

Phenotype
Several attempts to generate SpdS-deficient parasites were not successful (see RMgm-5259), despite the daily supplementation of Spermidine and Spermine to mice carrying the transfected parasites. To rule out any technical shortcoming or genetic accessibility in the inability to delete the PySpdS gene, we used a knock-in/knockout gene targeting strategy. In this strategy, the knock-in construct replaces the whole coding sequence or part of the coding sequence with an identical copy followed by eGFP sequence, and then followed by a constitutive 3' UTR. The knockout construct shares the same right homology arm as the knock-in construct, however, the left homology arm of the knockout construct was designed to delete or replace the gene with its own copy. The PCR genotyping confirms only the integration of the knock-in construct in the chromosomal locus (PySpdS-eGFP), however, the knockout construct for PySpdS did not integrate into the genome.

To find out the cellular localization of SpdS in mouse and mosquito stages of the parasites, blood-stages and sporozoites of the eGFP tagged PySpdS parasites were co-stained with the mitochondrial staining dye Mito-tracker Red. The eGFP signals of PySpdS were clearly co-localized with the mitochondrial marker dye Mito-tracker Red at both blood-stages and sporozoites. These results confirm the mitochondrial localization of the malaria parasite SpdS.

Additional information

Other mutants