DNA replication during intra-erythrocytic stages of human malarial parasite Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most virulent form of human malaria, has deservingly held the candidature of being one of the most studied human pathogens. Here we attempt an overview of the studies probing one of the important aspects of DNA metabolism, that is, DNA replication in this parasite, focusing on the relatively well-characterized core components of chromosomal DNA replication in the asexual intra-erythrocytic stage

The DNA replication apparatus in Plasmodium falciparum gametocytes

DNA synthesis in gametocytes takes place at two points; at the onset of gametocytogenesis where the nucleic acid content increases from lc to 1.8c prior to the formation of stage I gametocytes, and again 10 to 12 days later at the onset of gametogenesis when the mature male gametocyte (stage V) replicates its genome three times leading to the formation of eight haploid male gametes in less than 10 minutes. The aim of this study was to evaluate the status of the P. falciparum DNA replication apparatus during gametocytogenesis, most of which no significant DNA synthesis takes place, and gametogenesis in which a sudden burst of DNA replication takes place. The proteins studied in this project were DNA topoisomerase 1 and 11 (Topol and II), Replication factor C (Rfc) and Proliferating cell nuclear antigen (Pcna) of which P. falciparum homologous have been previously identified, isolated and characterised. Standard indirect immunofluorescence assays (IFA) carried out on unsynchronised in vitro cultivated P. falciparum (3D7A) using rabbit polyclonal antiserum raised against recombinant PfRfcl, PfRfc2, PfRfc3, PfPcna and PfTopoII showed that all five proteins are present throughout gametocytogenesis. All five proteins appear to be predominantly located within the nuclear region and at significantly higher levels in stage I and V gametocytes. However PfRfc2 levels appeared to be significantly higher only in stage I gametocytes and was distinctly absent from the nucleus of stage V gametocytes. Western blot analysis showed no significant changes in the levels of these proteins occurred during gametogenesis, with the exception of PfRfc2, which appear to increase immediately after activation and then gradually decrease as gametogenesis progressed.RT-PCR detected the presence of PfRFC2, PARFC3, PfPCNA, PfDNA POL5 and PiTOPO 1 transcripts in mature gametocytes before and after activation. However, PfRFCl and PfTOPO II transcripts were not detected in mature gametocytes either before or after activation. Similar results in protein and RNA analysis were obtained whether gametocytes were grown in AlbuMax or serum supplemented medium. In situ hybridisation using fluorescein-labelled PfRFCl, PfRFC2, PfDNA POL8, PfTOPO 1 and PfTOPO II gene fragments showed pockets of fluorescence on the peripheral regions of schizonts away from the nuclear region stained by DAPI. PfRFC3 and PfPCNA probes appeared to show fluorescence emanating from the nuclear region of schizonts. In conclusion, the localisation and unique expression pattern of PfRfc2 observed before and during gametogenesis, from that of the other DNA replication proteins and in particular, PfRfcl and PfRfc3, appears to imply a significant role for PfRfc2. Further studies need to be carried out in order to get to a better understanding of the role of PfRfc2 during gametogenesis

Global gene expression profiling of Plasmodium falciparum in response to the anti-malarial drug pyronaridine

<p>Abstract</p> <p>Background</p> <p>Pyronaridine (PN) and chloroquine (CQ) are structurally related anti-malarial drugs with primarily the same mode of action. However, PN is effective against several multidrug-resistant lines of <it>Plasmodium falciparum</it>, including CQ resistant lines, suggestive of important operational differences between the two drugs.</p> <p>Methods</p> <p>Synchronized trophozoite stage cultures of <it>P. falciparum </it>strain K1 (CQ resistant) were exposed to 50% inhibitory concentrations (IC₅₀) of PN and CQ, and parasites were harvested from culture after 4 and 24 hours exposure. Global transcriptional changes effected by drug treatment were investigated using DNA microarrays.</p> <p>Results</p> <p>After a 4 h drug exposure, PN induced a greater degree of transcriptional perturbation (61 differentially expressed features) than CQ (10 features). More genes were found to respond to 24 h treatments with both drugs, and 461 features were found to be significantly responsive to one or both drugs across all treatment conditions.</p> <p>Filtering was employed to remove features unrelated to primary drug action, specifically features representing genes developmentally regulated, secondary stress/death related processes and sexual stage development. The only significant gene ontologies represented among the 46 remaining features after filtering relate to host exported proteins from multi-gene families.</p> <p>Conclusions</p> <p>The malaria parasite's molecular responses to PN and CQ treatment are similar in terms of the genes and pathways affected. However, PN appears to exert a more rapid response than CQ. The faster action of PN may explain why PN is more efficacious than CQ, particularly against CQ resistant isolates. In agreement with several other microarray studies of drug action on the parasite, it is not possible, however, to discern mechanism of drug action from the drug-responsive genes.</p

RNA-Seq analysis of splicing in Plasmodium falciparum uncovers new splice junctions, alternative splicing and splicing of antisense transcripts.

Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5' GU-AG 3' and 5 new 5' GC-AG 3' junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite's transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite's transcriptome

Housekeeping and other metabolic functions of the Plasmodium plastid

The malaria parasite carries a plastid called the apicoplast that has been the subject of intense study in the last 15 years. Having originated from red-algal plastids, the apicoplast has lost its ability to photosynthesize, but carries out other essential functions such as type-II fatty acid synthesis, biosynthesis of haem and isoprenoid synthesis; the DOXP pathway for isoprenoid synthesis has recently been demonstrated to be the only pathway critical for parasite survival in the erythrocytic stage. The apicoplast also has a functional Suf system for assembly of (Fe–S) complexes on target proteins. The organelle has a 35 kb, double-stranded DNA genome that encodes a set of RNAs and proteins, the latter being translated from organellar mRNA by an active translation machinery, a major component of which is encoded by the nucleus. This article reviews current knowledge of housekeeping functions of the Plasmodium apicoplast and its (Fe–S) assembly system and discusses these components as sites for drug intervention against malaria

In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Malaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to <it>Plasmodium falciparum </it>infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the <it>Plasmodium falciparum </it>genome, we are still quite ignorant about <it>Plasmodium </it>mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription.</p> <p>Results</p> <p>A comprehensive directory of proteins reported to be potentially involved in <it>Plasmodium </it>transcriptional machinery was built from all <it>in silico </it>reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes), and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on <it>in silico </it>or on 2-yeast-hybrid experimental approaches is discussed.</p> <p>Conclusion</p> <p>This is the first attempt to build a comprehensive directory of potential transcription-associated proteins in <it>Plasmodium</it>. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of <it>Plasmodium falciparum </it>transcriptional regulation during the erythrocytic development.</p