A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development

Abstract

Plasmodium falciparum encodes a single calpain that has a distinct domain composition restricted to alveolates. To evaluate the potential of this protein as a drug target, we assessed its essentiality. Both gene disruption by double cross-over and gene truncation by single cross-over recombination failed. We were also unable to achieve allelic replacement by using a missense mutation at the catalytic cysteine codon, although we could obtain synonymous allelic replacement parasites. These results suggested that the calpain gene and its proteolytic activity are important for optimal parasite growth. To gain further insight into its biological role, we used the FKBP degradation domain system to generate a fusion protein whose stability in transfected parasites could be modulated by a small FKBP ligand, Shield1 (Shld1). We made a calpain-GFP-FKBP fusion through single cross-over integration at the endogenous calpain locus. Calpain levels were knocked down and parasite growth was greatly impaired in the absence of Shld1. Parasites were delayed in their ability to transition out of the ring stage and in their ability to progress to the S phase. Calpain is required for cell cycle progression in Plasmodium parasites and appears to be an attractive drug target. We have shown that regulated knockdowns are possible in P. falciparum and can be useful for evaluating essentiality and function.cell cycle ͉ cysteine protease ͉ essentiality ͉ inducible ͉ malaria

Atypical Mitogen-Activated Protein Kinase Phosphatase Implicated in Regulating Transition from Pre-S-Phase Asexual Intraerythrocytic Development of Plasmodium falciparum

“…A total of 100,000 cells were counted for each sample, and the data were analyzed using CFlow Plus software (BD Accuri). The cell cycle was determined by comparing the relative abundance of each developmental stage at each time point according to methods developed previously (33). The relative fold change was determined by calculating the fold increase in parasitemia between time zero and the endpoint.…”

Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag

“…The only conditional expression technology that is widely applicable in the parasite is the ddFKBP-based system (12)(13)(14), whose ligand is prohibitively expensive and toxic to the parasite (13). In this study, we tested the feasibility of using the DDD in P. falciparum.…”

“…Conditional expression systems such as the tetracycline-repressor system (11) have not been generally useful. FK506-binding protein degradation domain (ddFKBP)-based conditional expression was reported recently to work in the parasite (12)(13)(14). However, the small molecule that is used to stabilize the ddFKBP, Shield-1, is expensive and is somewhat toxic to the parasite at the concentrations required for protein stabilization (13).…”

Signal transduction in Plasmodium-Red Blood Cells interactions and in cytoadherence

“…Interestingly, calpains are cysteine proteases activated by calcium present in the host and the parasite. Phylogenetic analyses showed that many Plasmodium species has a calpain with homology on the N-terminal domain not found in the mammalian homology calpain (Russo et al 2009, Wu et al 2003. Interestingly the host calpain is involved in cytoskeletal remodeling (Goll et al 2003) and Plasmodium calpain-1 function has been related to parasite egress from erythrocyte whereas cysteine protease inhibitor blocked erythrocyte membrane disruption (Chandramohanadas et al 2009, Roiko andCarruthers 2009).…”

“…Interestingly the host calpain is involved in cytoskeletal remodeling (Goll et al 2003) and Plasmodium calpain-1 function has been related to parasite egress from erythrocyte whereas cysteine protease inhibitor blocked erythrocyte membrane disruption (Chandramohanadas et al 2009, Roiko andCarruthers 2009). Efforts to knockout the Plasmodium falciparum calpain was not successful, indicating the importance of this protease (Russo et al 2009). …”