Curcumin as an Adjunct Drug for Infectious Diseases.

Curcumin, by virtue of its ability to function as an immunomodulator, has the potential to serve as an adjunct drug to treat infectious diseases and provide long-term protection. The current need is to establish clinical trials with curcumin as an adjunct drug against specific infectious diseases.

Simultaneously targeting inflammatory response and parasite sequestration in brain to treat Experimental Cerebral Malaria.

Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15-20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. Migration of T cells and parasite-infected RBCs (pRBCs) into the brain are both necessary to precipitate the disease. We have been able to simultaneously target both these parameters of ECM. Curcumin alone was able to reverse all the parameters investigated in this study that govern inflammatory responses, CD8(+) T cell and pRBC sequestration into the brain and blood brain barrier (BBB) breakdown. But the animals eventually died of anemia due to parasite build-up in blood. However, arteether-curcumin (AC) combination therapy even after the onset of symptoms provided complete cure. AC treatment is a promising therapeutic option for HCM.

Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-(14)C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

Curcumin-arteether combination therapy of Plasmodium berghei-infected mice prevents recrudescence through immunomodulation.

Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha,beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2-3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INFγ and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INFγ levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2(-/-) and IL-10(-/-) animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR2(-/-) mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naïve animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to be tested for prevention of recrudescence in falciparum and relapse in vivax malaria.

Curcumin-artemisinin combination therapy for malaria.

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.

Curcumin for malaria therapy.

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.

Localization of ferrochelatase in Plasmodium falciparum.

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.

Delta-aminolevulinic acid dehydratase from Plasmodium falciparum: indigenous versus imported.

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.