Scientist
Division of Molecular and Structural Biology
Central Drug Research Institute,
P.O. Box No. 173, Chattar Manzil
Lucknow 226001

Subir Biswas, Research Fellow
Bijay Kumar, Research Fellow
Aiman Tanveer, Research Fellow
Ankit Gupta, Research Fellow
Kanika Kanchan, Project Assistant
Snober Mir, Post-doctoral Fellow

The primary research interest of my laboratory is to understand aspects of malaria apicoplast biology using molecular approaches. The apicoplast is a relict plastid of Plasmodium and is of interest as a site for drug intervention against malaria. We work on the following aspects:
(a) Understanding the mechanism of DNA replication and organization of the Plasmodium falciparum apicoplast.
Studies by our group have helped generate insights into the mechanism of replication of the apicoplast genome. We demonstrated that the ori of P.falciparum plDNA are localized within the inverted repeat region (IR) and that there are at least two initiation sites within each inverted repeat segment of the circular plDNA of P.falciparum suggestive of a four D-loop/bidirectional ori mechanism of DNA replication. Moreover, plDNA replication was shown to initiate with differential efficiencies at multiple sites within the inverted repeat region.

Figure: Signals observed for plDNA fragments using 5’-end labeled nascent DNA from replicating apicoplast DNA as probe.
Ori sequences were identified within the IR region.

The role of a P. falciparum nuclear-encoded bacterial histone-like protein (PfHU) in DNA compaction in the apicoplast has been revealed by studies in our laboratory. PfHU associates with apicoplast DNA and is expressed throughout the parasite’s intra-erythrocytic cycle. The protein displays a preference for supercoiled DNA and is capable of condensing E.coli nucleoids in vivo. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that
bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopy of PfHU-DNA complexes showed protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks.

These results have provided the first functional characterization of an apicomplexan HU protein and give additional evidence for red algal ancestry of the apicoplast.

Figure: AFM images of PfHUp- DNA complexes with increasing dimer/bp ratio. (A) DNA in the absence of protein.
(B) Dimer/bp ratio of 1:750. Stiffened DNA strands (panel i) as well as DNA bundles (panel ii) are shown.
(C) Dimer/bp ratio of 1:500. Formation of complexes with a small number of foci and extruding DNA loops (panel i),
a DNA bridge resulting in DNA looping (panel ii) and a nucleoprotein complex with a single focus (panel iii) are seen.

(b) Investigation of apicoplast ORFs, selected apicoplast-targeted proteins and their structure-function analysis.
Several proteins predicted to be involved in apicoplast-specific pathways are being studied. We attempt recombinant expression of these proteins and their functional analysis. The catalytic features of nuclear-encoded gyrase subunits have been analysed and the effect of novobiocin (a GyrB inhibitor) on apicoplast DNA replication and GyrB activity has indicated specific drug action on apicoplast GyrB. Novobiocin also causes parasite death in culture.

(c)Translation in the Plasmodium apicoplast.
Translation within the apicoplast is an attractive process for the discovery of novel antimalarials. Studies in our laboratory that localized the
apicoplast-encoded tufA gene product in the organelle and the effect of thiostrepton on EF-Tu levels provided evidence for active translation within the organelle. Under a collaborative project (project Mephitis) funded by European Commission’s Seventh Framework Programme, we are carrying out biochemical analysis of translation factors that function in the Plasmodium apicoplast.
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Figure: Immunofluorescence microscopy of a P.falciparum sporozoite using anti-EFTu Ab (red) and a mitochondrial dye (green)

An additional research area in the laboratory involves investigation of the role of human genetic variation and susceptibility to severe P.falciparum malaria in India. Drawing from SNP frequency data for a spectrum of Indian populations generated by the Indian Genome Variation Consortium, we analyse association
of specific variants with disease susceptibility in a falciparum endemic and a non-endemic region of India. SNPs in genes encoding adhesion and immune regulatory molecules are being studied in a case-control format.

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Figure: Frequency distribution of a TNF-a enhancer SNP in India and Pairwise FST analysis to study genetic differentiation among Indian populations (IGVC panel) using frequency data of SNPs previously associated with falciparum malaria.