Biomedical subjects
Publications and source records attributed to Govindarajan Padmanaban.
Artemisinin and curcumin show an additive interaction in killing Plasmodium falciparum in culture. In vivo, 3 oral doses of curcumin following a single injection of alpha,beta-arteether to Plasmodium berghei-infected mice are able to prevent recrudescence due to alpha,beta-arteether monotherapy and ensure almost 100% survival of the animals.
Malaria remains a major global health concern. New, inexpensive, and effective antimalarial agents are urgently needed. Here we show that curcumin, a polyphenolic organic molecule derived from turmeric, inhibits chloroquine-resistant Plasmodium falciparum growth in culture in a dose dependent manner with an IC(50) of approximately 5 microM. Additionally, oral administration of curcumin to mice infected with malaria parasite (Plasmodium berghei) reduces blood parasitemia by 80-90% and enhances their survival significantly. Thus, curcumin may represent a novel treatment for malarial infection.
Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (delta-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.
The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+. The enzyme is inhibited by Zn2+ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.