Single-stranded DNA binding protein from human malarial parasite Plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast

Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a ∼35 kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single–stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena

A Unique 45-Amino-Acid Region in the Toprim Domain of Plasmodium falciparum Gyrase B Is Essential for Its Activity▿ †

DNA gyrase is the only topoisomerase that can introduce negative supercoils into the DNA at the cost of ATP hydrolysis. Some but not all the steps of the topoisomerization reaction are understood clearly for both eukaryotic topoII and DNA gyrase. This study is an attempt to understand whether the B subunit of DNA gyrase binds to DNA directly, which may be central to the stimulation of its ATPase activity essential for gyrase function. We have dissected the Plasmodium falciparum gyrase B (PfGyrB) subunit to identify a 45-amino-acid region in the toprim domain that is responsible for its intrinsic DNA binding activity, DNA-stimulated ATPase activity, and DNA cleavage. We find that DNA has to enter through the ATP-operated clamp of PfGyrB to gain access to the DNA binding region. Furthermore, the rate of ATP hydrolysis of PfGyrB increases significantly with increasing DNA length, suggesting a possible communication between the ATPase domain and the DNA binding region that can account for its optimal ATPase activity. These results not only highlight the mechanism of GyrB action in the deadly human parasite P. falciparum but also provide meaningful insights into the current mechanistic model of DNA transport by gyrase during the topoisomerization reaction

Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth

Plasmodium falciparum sirtuin, PfSir2, contains Histone Deacetylase (HDAC) activity that may be central to the regulation of virulence gene expression in the parasites. Although a few reports have been published recently regarding in vitro and in vivo function of PfSir2, expression of the endogenous protein (c. 30 kDa) has not been shown yet. Here we report the presence of PfSir2 in the parasite at the protein level by specific antibodies. HDAC activity of PfSir2 can be inhibited by nicotinamide, a product of sirtuin reaction. Surprisingly, we find that nicotinamide also delays parasite growth significantly in culture. These findings further our knowledge on PfSir2 and raise the possibility of using an inexpensive agent like nicotinamide as an antimalarial in combination with other antiparasitic drugs

Potent antimalarial activity of Acriflavine in vitro and in vivo

Malaria continues to be a major health problem globally. There is an urgent need to find new antimalarials. Acriflavine (ACF) is known as an antibacterial agent and more recently as an anticancer agent. Here, we report that ACF inhibits the growth of asexual stages of both Chloroquine (CQ) sensitive and resistant strains of human malarial parasite, Plasmodium falciparum in vitro at nanomolar concentration. ACF clears the malaria infection in vivo from the bloodstreams of mice infected with Plasmodium berghei. Interestingly, ACF is accumulated only in the parasitized Red Blood Cells (RBCs) and parasite specific transporters may have role in this specific drug accumulation. We further show that ACF impairs DNA replication foci formation in the parasites and affects the enzymatic activities of apicoplast specific Gyrase protein. We thus establish ACF as a potential antimalarial amidst the widespread incidences of drug resistant Plasmodium strains

Potent Antimalarial Activity of Acriflavine <i>In Vitro</i> and <i>In Vivo</i>

Malaria continues to be a major health problem globally. There is an urgent need to find new antimalarials. Acriflavine (ACF) is known as an antibacterial agent and more recently as an anticancer agent. Here, we report that ACF inhibits the growth of asexual stages of both chloroquine (CQ) sensitive and resistant strains of human malarial parasite, <i>Plasmodium falciparum in vitro</i> at nanomolar concentration. ACF clears the malaria infection <i>in vivo</i> from the bloodstreams of mice infected with <i>Plasmodium berghei</i>. Interestingly, ACF is accumulated only in the parasitized red blood cells (RBCs) and parasite specific transporters may have role in this specific drug accumulation. We further show that ACF impairs DNA replication foci formation in the parasites and affects the enzymatic activities of apicoplast specific Gyrase protein. We thus establish ACF as a potential antimalarial amidst the widespread incidences of drug resistant <i>Plasmodium</i> strains

() Far-UV circular dichroism spectra of wild-type and different mutant variants of the helicase

<p><b>Copyright information:</b></p><p>Taken from "The domain structure of DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function"</p><p></p><p>Nucleic Acids Research 2007;35(9):2861-2874.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1888833.</p><p>© 2007 The Author(s)</p> CD spectra were measured for each protein in a 2 mm path length quartz cell and data were collected at 1.0 nm wavelength resolution. The arrowheads indicate the CD spectra of the respective protein. () Intrinsic fluorescence spectra of wild-type and different deletion mutants. The fluorescence emission spectra of wild-type and different mutant forms of HpDnaB (as indicated) were recorded from 300 to 400 nm at 25°C. The excitation wavelength was 278 nm and data were collected at 0.5 nm wavelengths resolution. The arrowheads indicate the CD spectra of the respective protein

Electron microscopic observation and analysis of HpDnaBWt, DelN2, DelN3 and DelC1

<p><b>Copyright information:</b></p><p>Taken from "The domain structure of DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function"</p><p></p><p>Nucleic Acids Research 2007;35(9):2861-2874.</p><p>Published online 11 Apr 2007</p><p>PMCID:PMC1888833.</p><p>© 2007 The Author(s)</p> Above four proteins were processed for electron microscopy as described in the materials and methods and individual sample was scanned under a Morgagni 268 transmission electron microscope at 80 kV voltage. More than hundred images were captured in each case and the raw images were processed using IMAGIC software. The left panel in each pair shows the unprocessed image and the right panel shows the processed image. HpDnaBWt was found in both C6 and C3 conformations whereas DelN2 was found only in C3 conformations. Bars in the panels are equivalent to 10 nm