Mutant/mutation
The mutant expresses a mutated form of DDI1: a fusion of P. falciparum DDIMyc with HA-tagged mutant E. coli DHFR (cDDHA). cDD binds trimethoprim and is stable, but undergoes proteasomal degradation in the absence of trimethoprim

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.10.29.466443.

Protein (function)
PF3D7_1409300 DDI1 was first discovered as one of the over expressed proteins upon treatment of Saccharomyces cerevisiae with DNA damaging agents, hence, it was named as the DNA damage inducible 1 protein (DDI1). Deletion analysis demonstrated that both UBL and RVP domains of S. cerevisiae DDI1 (ScDDI1) are necessary for inhibition of protein secretion. DDI1 proteins are conserved in eukaryotes and involved in a variety of cellular processes, including proteasomal degradation of specific proteins and DNA-protein crosslink repair. All DDI1 proteins contain ubiquitin-like (UBL) and retroviral aspartyl protease (RVP) domains, and some also contain ubiquitin-associated (UBA) domain, which mediate distinct activities of these proteins. The DDI1 proteins of Plasmodium and other Apicomplexan parasites vary in domain architecture, share UBL and VP domains, and the majority of proteins contain the UBA domain.

Phenotype
DDI1 is essential for parasite development. Expression of DDI1 in all the major parasite stages suggests its importance for parasite development. Hence, we attempted to knock-out the PbDDI1 gene for investigation of its functions during parasite development. However, multiple knock-out attempts were unsuccessful.

We next employed a conditional knock-down approach in P. berghei by replacing the wild type PbDDI1 coding region with PfDDIMyc/cDDHA coding sequence, which would express fusion of PfDDIMyc with HA-tagged mutant E. coli DHFR (cDDHA). cDD binds trimethoprim and is stable, but undergoes proteasomal degradation in the absence of trimethoprim, thereby, causing a knock-down effect. The replacement of wild type PbDDI1 with PfDDIMyc/cDDHA and expression of DDIMyc/cDDHA fusion protein were successful, which confirmed that PbDDI1 and PfDDI1 are functionally conserved.

The knock-down parasites (PbKD) showed reduction in DDIMyc/cDDHA protein level in the absence of trimethoprim compared to that in the presence of trimethoprim, indicating the knock-down effect. PbKD parasites grew similar to wild type parasites in the presence of trimethoprim, but showed drastically reduced growth in the absence of trimethoprim and eventually disappeared. In the presence of trimethoprim, PbKD-infected mice had to be euthanized, whereas in the absence of trimethoprim, the parasitemia barely reached to 0.5% and the infection was self-limiting. Withdrawal of trimethoprim from PbKD-infected mice about 5% parasitemia also resulted in complete clearance of parasites.

Additional information
Evidence is shown that:
- Plasmodium DDI1 is expressed in all major parasite stages
- DDI knock-down enhanced drug sensitivity of parasites
- DDI1 associates with chromatin and DNA-protein crosslinks
- DDI1 interacts with pre-mRNA-processing factor 19

Other mutants