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SUMMARY OF DOCTORAL WORK Thesis Title: Studies with antioxidant system enzymes to explore the molecular mechanism of arteether resistance in Plasmodium vinckei. Malaria parasite is highly susceptible to oxidative stress during its intra-erythrocytic stage development and parasites are highly adapted to cope with this stress via elaborate redox and antioxidant defense system. Artemisinin derivatives act against the intra erythrocytic blood stages via free-radical generation. Since redox mechanism is a common mechanism to cope with this oxidative stress caused by free radicals, there is a strong possibility of involvement of stress related proteins in resistance to artemisinin derivatives. Hence, the present study was conceptualized to understand the mechanism of resistance to artemisinin derivative arteether, employing experimental rodent malaria model. Form the parent Arteether sensitive Plasmodium vinckei (PvAS) strain, the derived strain showing >48 fold resistance to arteether was selected by sub curative drug pressure and was referred to as Arteether resistant Plasmodium vinckei (PvAR) strain. Both the strains were maintained in vivo in Swiss mice by blood induced sequential passages. Cell free parasite preparations were obtained for both these strains after passage of the pooled infected blood through CF11 column followed by saponization. These preparations have been used for RNA isolation, cDNA synthesis and genomic DNA isolation required for molecular cloning and characterization studies. The study demonstrated that administration of arteether to Plasmodium vinckei, a rodent malaria parasite, resulted in increased oxidative stress. However, in arteether resistant P.vinckei parasites, treatment with arteether results in decreased generation of reactive oxygen species and increased expression of antioxidant system genes reflecting their increased capability of tolerating stressful conditions. A total of 25 genes from five different families of antioxidant system were selected for the study. Among them, the highly expressed antioxidant genes in resistant parasites were Superoxide dismutase1 (SOD1), Glutathione reductase (GR), Protein disulphide isomerase 11 (PDI11) and Thioredoxin 2 (Trx2). Comparison of gene sequences of these four enzymes from sensitive and resistant parasites did not show any mutation in SOD1 and PDI11. However GR sequence for resistant parasite showed 15 mutations out of which two mutations (S133N and N291I) were found to be significant. Likewise thioredoxin sequence showed four mutations, out of which two mutations (S35L and T78C) were regarded important for further investigations. The superoxide dismutase family proteins are of enormous attention and extraordinary values with respect to malaria parasites due to their residence within the host red blood cells, place where excessive production of reactive oxygen species is endorsed through hemoglobin degradation. The present study has identified, sequenced and biochemically characterized a putative iron dependent superoxide dismutase from rodent malaria parasite P. vinckei (PvSOD1). The sequence analyses of PvSOD1 revealed a close similarity with other iron dependent superoxide dismutase. Cross-linking, Native-PAGE and FPLC gel filtration analyses documented that PvSOD1 exists as a dimer in solution, a common feature shared by other Fe-SODs. PvSOD1 is highly expressed during ring and trophozoite stages of parasite compared to that of schizont stage as evident through RT-PCR and immunofluorescence studies. Enzymatic activity of recombinant PvSOD1 was validated using conventional zymogram analyses and xanthine - xanthine oxidase system. Under optimal conditions recombinant PvSOD1 is highly active and catalyze the dismutation of superoxide radicals comparable to that of other bonafide superoxide dismutase. Under excessive oxidative stress, protein S-glutathionylation of PvSOD1 protects the enzyme from reversible thiol modification, thereby protecting parasite SOD1 from inactivation under intense oxidative stress conditions. In this regard we have studied the presence of glutathionylation of Fe-SOD1 from rodent malaria parasite P.vinckei (PvSOD1) and elucidated its impact on protein function. The parasite encoded SOD1 reacts well with the anti-GSH antibody suggesting the presence of glutathionylation. PvSOD1 also undergoes S-glutathionylation during its overproduction in bacterial cells in a concentration dependent manner when treated with H2O2, suggesting a physiological role under excessive oxidative stress. Also the effect of various oxidizing and reducing agents on PvSOD1 glutathionylation was studied by western immuno- blotting and MALDI - Mass analysis. The present study has concluded that arteether exposure to P.vinckei parasites results in increased oxidative stress. However, in arteether resistant P.vinckei parasites, treatment with arteether results in decreased generation of reactive oxygen species and increased expression of antioxidant system genes reflecting their increased capability of tolerating stressful conditions. Among the highly expressed antioxidant genes in PvAR parasites were SOD1, GR, PDI11 and Trx2. Further studies with these four genes revealed that only Trx2 from arteether resistant parasite has mutation near the active site CxxC which results in formation of new domain CxxxCxxC, a characteristic feature of S-adenosyl methionine family protein. Furthermore, under excessive oxidative stress, protein S-glutathionylation of PvSOD1 protects the enzyme from reversible thiol modification, thereby protecting parasite SOD1 from inactivation under intense oxidative stress conditions. PvSOD1 is an important enzyme of the antioxidant defense pathway in P.vinckei protecting the parasite under oxidative burst and is a potential drug targetfor discovering new antimalarials. It is proposed that an assay system employing recombinant PvSOD1 can identify specific inhibitors for development as potential agents for improved chemotherapy against malaria. ACADEMIC PROJECT DURING MASTER'S Project Title: Expression and purification of Sodium Stibogluconate resistant gene of (ssgr- 1) of Leismania donovani. Organization: CSIR-CDRI, Lucknow, Summary: Construct TOPO-T7-ssgr-1 having gene was transformed in E.coli BL21-DE3 cells. The gene was expressed as His tag fusion protein and partially purified by Ni-NTA chromatography. Also suitability of two methods for isolation of genomic DNA (phenol: chloroform and kit method) for southern analyses were done. This project was helpful in characterization of antimony resistance gene and in drug target development.
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