Defining species specific genome differences in malaria parasites

<p>Abstract</p> <p>Background</p> <p>In recent years a number of genome sequences for different <it>plasmodium </it>species have become available. This has allowed the identification of numerous conserved genes across the different species and has significantly enhanced our understanding of parasite biology. In contrast little is known about species specific differences between the different genomes partly due to the lower sequence coverage and therefore relatively poor annotation of some of the draft genomes particularly the rodent malarias parasite species.</p> <p>Results</p> <p>To improve the current annotation and gene identification status of the draft genomes of <it>P. berghei</it>, <it>P. chabaudi </it>and <it>P. yoelii</it>, we performed genome-wide comparisons between these three species. Through analyses via comparative genome hybridizations using a newly designed pan-rodent array as well as in depth bioinformatics analysis, we were able to improve on the coverage of the draft rodent parasite genomes by detecting orthologous genes between these related rodent parasite species. More than 1,000 orthologs for <it>P. yoelii </it>were now newly associated with a <it>P. falciparum </it>gene. In addition to extending the current core gene set for all plasmodium species this analysis also for the first time identifies a relatively small number of genes that are unique to the primate malaria parasites while a larger gene set is uniquely conserved amongst the rodent malaria parasites.</p> <p>Conclusions</p> <p>These findings allow a more thorough investigation of the genes that are important for host specificity in malaria.</p

Quantitative protein expression profiling reveals extensive post-transcriptional regulation and post-translational modifications in schizont-stage malaria parasites

A quantitative time-course analysis of protein abundance for Plasmodium falciparum schizonts using two-dimensional differential gel electrophoresis reveals significant post-transcriptional regulation

Characterization of the repertoire diversity of the Plasmodium falciparum stevor multigene family in laboratory and field isolates

BACKGROUND: The evasion of host immune response by the human malaria parasite Plasmodium falciparum has been linked to expression of a range of variable antigens on the infected erythrocyte surface. Several genes are potentially involved in this process with the var, rif and stevor multigene families being the most likely candidates and coding for rapidly evolving proteins. The high sequence diversity of proteins encoded by these gene families may have evolved as an immune evasion strategy that enables the parasite to establish long lasting chronic infections. Previous findings have shown that the hypervariable region (HVR) of STEVOR has significant sequence diversity both within as well as across different P. falciparum lines. However, these studies did not address whether or not there are ancestral stevor that can be found in different parasites. METHODS: DNA and RNA sequences analysis as well as phylogenetic approaches were used to analyse the stevor sequence repertoire and diversity in laboratory lines and Kilifi (Kenya) fresh isolates. RESULTS: Conserved stevor genes were identified in different P. falciparum isolates from different global locations. Consistent with previous studies, the HVR of the stevor gene family was found to be highly divergent both within and between isolates. Importantly phylogenetic analysis shows some clustering of stevor sequences both within a single parasite clone as well as across different parasite isolates. CONCLUSION: This indicates that the ancestral P. falciparum parasite genome already contained multiple stevor genes that have subsequently diversified further within the different P. falciparum populations. It also confirms that STEVOR is under strong selection pressure

De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection

Immunodeficient mouse–human chimeras provide a powerful approach to study host specific pathogens like Plasmodium (P.) falciparum that causes human malaria. Existing mouse models of P. falciparum infection require repeated injections of human red blood cells (RBCs). In addition, clodronate lipsomes and anti-neutrophil antibodies are injected to suppress the clearance of human RBCs by the residual immune system of the immunodeficient mice. Engraftment of NOD-scid Il2rg[superscript -/-] mice with human hematopoietic stem cells leads to reconstitution of human immune cells. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, human RBC reconstitution is generally poor or undetectable. The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. Delivery of plasmid DNA encoding human erythropoietin and interleukin-3 into humanized mice by hydrodynamic tail-vein injection resulted in significantly enhanced reconstitution of erythrocytes. With this improved humanized mouse, here we show that P. falciparum infects de novo generated human RBCs, develops into schizonts and causes successive reinvasion. We also show that different parasite strains exhibit variation in their ability to infect these humanized mice. Parasites could be detected by nested PCR in the blood samples of humanized mice infected with P. falciparum K1 and HB3 strains for 3 cycles, whereas in other strains such as 3D7, DD2, 7G8, FCR3 and W2mef parasites could only be detected for 1 cycle. In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles. The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection. Compared to existing mouse models, our model generates human RBCs de novo and does not require the treatment of mice with immunomodulators.Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

tRNA epitranscriptomics and biased codon are linked to proteome expression in

Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites.Singapore. National Research FoundationSingapore-MIT Alliance (Graduate Fellowship

Structural Determination of Functional Units of the Nucleotide Binding Domain (NBD94) of the Reticulocyte Binding Protein Py235 of Plasmodium yoelii

Invasion of the red blood cells (RBC) by the merozoite of malaria parasites involves a large number of receptor ligand interactions. The reticulocyte binding protein homologue family (RH) plays an important role in erythrocyte recognition as well as virulence. Recently, it has been shown that members of RH in addition to receptor binding may also have a role as ATP/ADP sensor. A 94 kDa region named Nucleotide-Binding Domain 94 (NBD94) of Plasmodium yoelii YM, representative of the putative nucleotide binding region of RH, has been demonstrated to bind ATP and ADP selectively. Binding of ATP or ADP induced nucleotide-dependent structural changes in the C-terminal hinge-region of NBD94, and directly impacted on the RBC binding ability of RH.In order to find the smallest structural unit, able to bind nucleotides, and its coupling module, the hinge region, three truncated domains of NBD94 have been generated, termed NBD94(444-547), NBD94(566-663) and NBD94(674-793), respectively. Using fluorescence correlation spectroscopy NBD94(444-547) has been identified to form the smallest nucleotide binding segment, sensitive for ATP and ADP, which became inhibited by 4-Chloro-7-nitrobenzofurazan. The shape of NBD94(444-547) in solution was calculated from small-angle X-ray scattering data, revealing an elongated molecule, comprised of two globular domains, connected by a spiral segment of about 73.1 A in length. The high quality of the constructs, forming the hinge-region, NBD94(566-663) and NBD94(674-793) enabled to determine the first crystallographic and solution structure, respectively. The crystal structure of NBD94(566-663) consists of two helices with 97.8 A and 48.6 A in length, linked by a loop. By comparison, the low resolution structure of NBD94(674-793) in solution represents a chair-like shape with three architectural segments.These structures give the first insight into how nucleotide binding impacts on the overall structure of RH and demonstrates the potential use of this region as a novel drug target

Comparative Heterochromatin Profiling Reveals Conserved and Unique Epigenome Signatures Linked to Adaptation and Development of Malaria Parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium

Changes in Parasite Virulence Induced by the Disruption of a Single Member of the 235 kDa Rhoptry Protein Multigene Family of Plasmodium yoelii

Invasion of the erythrocyte by the merozoites of the malaria parasite is a complex process involving a range of receptor-ligand interactions. Two protein families termed Erythrocyte Binding Like (EBL) proteins and Reticulocyte Binding Protein Homologues (RH) play an important role in host cell recognition by the merozoite. In the rodent malaria parasite, Plasmodium yoelii, the 235 kDa rhoptry proteins (Py235) are coded for by a multigene family and are members of the RH. In P. yoelii Py235 as well as a single member of EBL have been shown to be key mediators of virulence enabling the parasite to invade a wider range of host erythrocytes. One member of Py235, PY01365 is most abundantly transcribed in parasite populations and the protein specifically binds to erythrocytes and is recognized by the protective monoclonal antibody 25.77, suggesting a key role of this particular member in virulence. Recent studies have indicated that overall levels of Py235 expression are essential for parasite virulence. Here we show that disruption of PY01365 in the virulent YM line directly impacts parasite virulence. Furthermore the disruption of PY01365 leads to a reduction in the number of schizonts that express members of Py235 that react specifically with the mcAb 25.77. Erythrocyte binding assays show reduced binding of Py235 to red blood cells in the PY01365 knockout parasite as compared to YM. While our results identify PY01365 as a mediator of parasite virulence, they also confirm that other members of Py235 are able to substitute for PY01365

Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

<p>Abstract</p> <p>Background</p> <p>Artemisinin resistance in <it>Plasmodium falciparum </it>malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates.</p> <p>Results</p> <p>In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the <it>P. falciparum </it>intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype.</p> <p>Conclusions</p> <p>The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes <it>in vivo</it>.</p