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To counteract this increase in stress; parasite has to produce anti-oxidant enzymes or increase the expression of membrane channels to import anti-oxidant molecules (such as GSH from RBC) from the RBC cytosol. RIO-2 kinases are known to participate in ribosome maturation and proposed to have an indirect role in protein production, required to manage the oxidative stress (LaRonde- LeBlanc and Wlodawer 2005). To test the relevance of PFD0975w in the parasite biology during exogenous stress, we have challenged the parasites with two different kinds of stress: (1) starvation, such as depletion of glucose from culture medium, and (2) anti-malarial drug pressure. The pattern of PFD0975w signal changes in glucose starved parasite (Figure 2.6).
The localization pattern of PFD0975w during the ring stage of treated parasite gives uniform level of expression throughout the parasite cytosol. It is similar to that of the ring stage of untreated parasite (Figure 2.5C).
In the mature stages of the parasite such as trophozoite and schizont, the protein appears to be expressed in a punctuate fashion with condensed spots of PFD0975w signal within cytoplasm.
Glucose starvation thus shifts the uniformity of PFD0975w expression into localized cellular spots.
The probable explanation for the observed pattern is that the ring stage parasites are rich in nutrients and hence better ability to manage stress caused by glucose starvation. As a result, PFD0975w expression pattern is least responsive to exogenous stress during the ring stage. Transition of ring stage into trophozoite and schizont, nutrients deplete within the infected cell with lesser resources to manage stress. The marked change in the localization pattern of PFD0975w in trophozoite and schizont could be a means to overcome stress due to starvation. Parasite treated with 0.5 µg/ml chloroquine; exhibit a marked overall down-regulation of PFD0975w expression during the RBC stages (Figure 2.7). The observed pattern reflects oxidative stress generated within the parasite as a result of anti-malarial drug pressure.
Figure 2.7: Immunolocalization of PFD0975w in Plasmodium falciparum exposed to exogenous stress: Exposure to 0.5 µg/ml (IC50) of antimalarial agent chloroquine.
intent of cloning and over expression of the RIO-2 kinase from the malaria parasite is due to the belief that this kinase might play an essential role in the parasite’s life cycle too. Bacterial expression system is chosen because of its simplicity, abundance of over-expressed protein within the bacterial cell, cost-effectiveness and yield of the purification process. The initial attempt was to clone the native PfRIO-2 kinase gene from Plasmodium falciparum using PCR amplification. The native gene failed to express in bacterial system due to codon bias between prokaryotic and eukaryotic protein
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synthesis machinery. To circumvent the expression problem, a codon optimized synthetic version of PfRIO2- kinase was commercially synthesized. This synthetic version (PFD0975ws) of the gene was designed for high level expression in the bacterial system and consisted of the winged helix domain (residues 1-88) and the protein kinase domain (residues 89-270) of the native protein.
The next major challenge was the appearance of the protein in insoluble fraction. Solubility optimization experiments were conducted to see the effect of different host bacterial strains, media supplementation, buffer pH, inducer concentration, induction time and temperature on the yield of soluble PFD0975ws. The results revealed that the highest yield of soluble protein was obtained when Rosetta-gami™ host strains were used to express the protein. Rosetta-gami™ host cells, from Novagen are derivatives of Origami™, that combine the enhanced disulfide bond formation resulting from trxB/gor mutations with enhanced expression of eukaryotic protein that contain codons rarely used in E. coli. This strain supplies tRNAs for AGG, AGA, AUA, CUA, CCC, and GGA on a compatible chloramphenicol-resistant plasmid. The hexa-histidine tagged recombinant PfRIO-2 kinase was purified using immobilized metal affinity chromatography and its native molecular weight is estimated to be ~ 35 KDa using gel image analyses. Antibodies were generated against PFD0975w in rabbit model and the purified polyclonal antibody was used to study the changes in expression pattern of PFD0975w during the parasite’s asexual stages in the presence and absence of exogenous cellular stress. Studies on Saccharomyces cerevisiae and cancer cell lines have shown that exogenous cellular stress like glucose starvation results in increased phosphorylation of specific protein kinases along with the production of ROS (Graham, Tahmasian et al. 2012, Braun, Vaga et al.
2014). Studies have also correlated the cellular stress in Plasmodium falciparum with the increased phosphorylation of eIF2α kinases like PK4 (Zhang, Mishra et al. 2012). The shift in the observed expression of PFD0975w during glucose starvation and in the presence of antimalarial chloroquine indicates a correlation between PFD0975w and parasite stress response. The results of our current work juxtaposed with our previous findings (Nag, Prasad et al. 2012, Trivedi and Nag 2012, Nag, Chouhan et al. 2013), strongly suggests PFD0975w as a key player in the cellular signal transduction events during growth as well as under stress conditions. The expression pattern of this protein kinase is strongly governed by the stage of growth as well as the microenvironment and stress factors involved which surround the parasite. The results indicate that PFD0975w might be an essential cellular gene in Plasmodium falciparum and can be an attractive drug target, opening new avenues for antimalarial chemotherapy.