Pundi N. Rangarajan

Haem as a multifunctional regulator

Haem has long been known as the prosthetic group of haemoproteins such as haemoglobin, catalase and the cytochromes. Its biosynthesis is regulated by feedback mechanisms that ensure its adequate production but prevent its overaccumulation, which is highly deleterious as diseases such as porphyrias attest. However, recent years have seen rapid strides in our understanding of how haem (or more accurately haemin, its oxidized form) itself acts as an intracellular regulator of a variety of other metabolic pathways for systems that utilize oxygen.

Curcumin-Artemisinin Combination Therapy for Malaria

ABSTRACT 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 α,β-arteether to Plasmodium berghei-infected mice are able to prevent recrudescence due to α,β-arteether monotherapy and ensure almost 100% survival of the animals.

Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target.

The parasite Plasmodium berghei imports the enzyme δ-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-ΔNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-ΔNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death.

Involvement of δ-aminolaevulinate synthase encoded by the parasite gene in de novo haem synthesis by Plasmodium falciparum

The malaria parasite can synthesize haem de novo. In the present study, the expression of the parasite gene for delta-aminolaevulinate synthase (Pf ALAS ) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture. The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage. Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa. The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P. falciparum and growth in culture. The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated. The import is blocked by haemin. On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target. The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis. The Pf ALAS gene is functional and vital for parasite haem synthesis and parasite survival.

δ-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 δ-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.

Protective efficacy of a plasmid DNA encoding Japanese encephalitis virus envelope protein fused to tissue plasminogen activator signal sequences: studies in a murine intracerebral virus challenge model

We report the construction of chimeric DNA vaccine vectors in which secretory signal sequence derived from tissue plasminogen activator (TPA) was fused to the full length (pCMVTE) or 398 amino terminal amino acids (pCMVTdeltaE) of Japanese encephalitis virus (JEV) envelope (E) protein. Transfection studies indicate that E protein expressed from pCMVTdeltaE-transfected cells but not pCMVTE-transfected cells is secreted into the culture medium. Analysis of the potency of various DNA vaccine constructs in a murine intracerebral (i.c.) JEV challenge model indicates that pCMVTdeltaE confers the highest level (71%) of protection. Immunization with pCMVTdeltaE induces a mixed Th1 and Th2 T helper cell response while immunization with plasmids encoding nonsecretory forms of E protein induces a Th1 T helper response. Only low levels (<1:20) of virus neutralizing antibody titres were observed in DNA vaccinated mice which did not increase further after i.c. JEV challenge. Thus, immunization with a plasmid encoding secretory E protein results in an altered cytokine response and better protection against i.c. JEV challenge than that conferred by immunization with plasmids encoding nonsecretory forms of E protein. We also demonstrate that unlike peripheral JEV challenge, i.c. JEV challenge does not result in an increase in anamnestic antibody response suggesting that other components of immune system such as cytotoxic T cells and T helper cells contribute to protection against i.c. JEV challenge of DNA vaccinated mice.

Immunization with plasmid DNA encoding the envelope glycoprotein of Japanese Encephalitis virus confers significant protection against intracerebral viral challenge without inducing detectable antiviral antibodies

A plasmid DNA construct, pCMXENV encoding the envelope (E) glycoprotein of Japanese Encephalitis virus (JEV), was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. The ability of pCMXENV to protect mice from lethal JEV infection was evaluated using an intracerebral (i.c.) JEV challenge model. Several independent immunization and JEV challenge experiments were carried out and the results indicate that 51 and 59% of the mice are protected from lethal i.c. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. None of the mice immunized with the vector DNA (pCMX) survived in any of these experiments. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-gamma (IFN-gamma) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Th1 sub-type. In conclusion, our results demonstrate that immunization with a plasmid DNA expressing an intracellular form of JEV E protein confers significant protection against i.c. JEV challenge even in the absence of detectable antiviral antibodies.

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.

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Delta)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.

Preexposure efficacy of a novel combination DNA and inactivated rabies virus vaccine

Several strategies are being examined to enhance the potency of DNA rabies vaccine (DRV) so that it can be used for both prophylaxis and postexposure therapy of rabies. In this study, we report a novel combination rabies vaccine (CRV) containing a low dose of cell culture-derived inactivated rabies virus vaccine and DRV. Mice immunized with CRV develop higher levels of rabies virus-neutralizing antibodies (RVNA) than those immunized with DRV and are completely protected against peripheral as well as intracerebral rabies virus challenge. The quantity of inactivated rabies virus vaccine required for enhancing the potency of DRV can be 625-fold lower than that of a standard dose of inactivated rabies virus vaccine. CRV induces higher levels of RVNA than DRV in cattle as well. Thus, we demonstrate for the first time that co-inoculation of DNA vaccine and a low dose of inactivated virus vaccine can be developed into a novel cost-effective vaccination strategy for combating rabies in particular, and infectious diseases in general.