Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites.
Plasmodium falciparum; antigenic variation; genome conformation capture; 3C; HiC
Intraerythrocytic development of the human malaria parasite Plasmodium falciparum appears as a continuous flow through growth and proliferation. To develop a greater understanding of the critical regulatory events, we utilized piggyBac insertional mutagenesis to randomly disrupt genes. Screening a collection of piggyBac mutants for slow growth, we isolated the attenuated parasite C9, which carried a single insertion disrupting the open reading frame (ORF) of PF3D7_1305500. This gene encodes a protein structurally similar to a mitogen-activated protein kinase (MAPK) phosphatase, except for two notable characteristics that alter the signature motif of the dual-specificity phosphatase domain, suggesting that it may be a low-activity phosphatase or pseudophosphatase. C9 parasites demonstrated a significantly lower growth rate with delayed entry into the S/M phase of the cell cycle, which follows the stage of maximum PF3D7_1305500 expression in intact parasites. Genetic complementation with the full-length PF3D7_1305500 rescued the wild-type phenotype of C9, validating the importance of the putative protein phosphatase PF3D7_1305500 as a regulator of pre-S-phase cell cycle progression in P. falciparum.
Studies on DNA from pathogenic organisms, within clinical samples, are often complicated by the presence of large amounts of host, e.g., human DNA. Isolation of pathogen DNA from these samples would improve the efficiency of next-generation sequencing (NGS) and pathogen identification. Here we describe a solution-based hybridisation method for isolation of pathogen DNA from a mixed population. This straightforward and inexpensive technique uses probes made from whole-genome DNA and off-the-shelf reagents.
In this study, Escherichia coli DNA was successfully enriched from a mixture of E.coli and human DNA. After enrichment, genome coverage following NGS was significantly higher and the evenness of coverage and GC content were unaffected. This technique was also applied to samples containing a mixture of human and Plasmodium falciparum DNA. The P.falciparum genome is particularly difficult to sequence due to its high AT content (80.6%) and repetitive nature. Post enrichment, a bias in the recovered DNA was observed, with a poorer representation of the AT-rich non-coding regions. This uneven coverage was also observed in pre-enrichment samples, but to a lesser degree. Despite the coverage bias in enriched samples, SNP (single-nucleotide polymorphism) calling in coding regions was unaffected and the majority of samples had over 90% of their coding region covered at 5× depth.
This technique shows significant promise as an effective method to enrich pathogen DNA from samples with heavy human contamination, particularly when applied to GC-neutral genomes.
AT-rich DNA; clinical samples; E.coli; enrichment; host DNA contamination; in-solution hybridisation; next generation sequencing; P.falciparum
Purpose. To investigate mesh coating modalities with autologous blood components in a recently developed in vitro test system for biocompatibility assessment of alloplastic materials. Materials and Methods. Seven different mesh types, currently used in various indications, were randomly investigated. Meshes were coated prior to cultivation with autologous peripheral blood mononuclear cells (PBMCs), platelets, and blood plasma. Pretreated meshes were incubated over 6 weeks in a minced tissue assay, representative for fibroblasts, muscle cells, and endothelial cells originating from 10 different patients. Adherence of those tissues on the meshes was microscopically investigated and semiquantitatively assessed using a previously described scoring system. Results. Coating with peripheral blood mononuclear cells did not affect the adherence score, whereas coating with platelets and blood plasma increased the score suggesting improved biocompatibility in vitro. The previous ranking of native meshes remained consistent after coating. Conclusion. Plasma coating of meshes improves their biocompatibility score in a novel in vitro test system.
Methods to reliably assess the accuracy of genome sequence data are lacking. Currently completeness is only described qualitatively and mis-assemblies are overlooked. Here we present REAPR, a tool that precisely identifies errors in genome assemblies without the need for a reference sequence. We have validated REAPR on complete genomes or de novo assemblies from bacteria, malaria and Caenorhabditis elegans, and demonstrate that 86% and 82% of the human and mouse reference genomes are error-free, respectively. When applied to an ongoing genome project, REAPR provides corrected assembly statistics allowing the quantitative comparison of multiple assemblies. REAPR is available at http://www.sanger.ac.uk/resources/software/reapr/.
Genome assembly; validation; evaluation
Genome projects now produce draft assemblies within weeks thanks to advanced high-throughput sequencing technologies. For milestone projects like E. coli or H. sapiens, teams of scientists were employed to manually curate and finish these genomes to a high standard. Nowadays, this is not feasible for most projects and the quality of genomes is generally of a much lower standard. This protocol describes software (PAGIT, post-assembly genome-improvement toolkit) to improve the quality of draft genomes. It offers flexible functionality to close gaps in scaffolds, correct base errors in the consensus sequence, and to exploit reference genomes (if available) for improving scaffolding and generating annotations. The protocol is most accessible for bacterial and small Eukaryotic genomes (up to 300 Mb), such as pathogenic bacteria, malaria and parasitic worms. Applying PAGIT to an E. coli assembly takes approximately 24 hours: it doubles the average contig size and annotates over 4300 gene models.
Next generation sequencing; automatic finishing; gap closing; genome annotation; contig ordering
The cost of whole-genome sequencing (WGS) is decreasing rapidly as next-generation sequencing technology continues to advance, and the prospect of making WGS available for public health applications is becoming a reality. So far, a number of studies have demonstrated the use of WGS as an epidemiological tool for typing and controlling outbreaks of microbial pathogens. Success of these applications is hugely dependent on efficient generation of clean genetic material that is free from host DNA contamination for rapid preparation of sequencing libraries. The presence of large amounts of host DNA severely affects the efficiency of characterizing pathogens using WGS and is therefore a serious impediment to clinical and epidemiological sequencing for health care and public health applications. We have developed a simple enzymatic treatment method that takes advantage of the methylation of human DNA to selectively deplete host contamination from clinical samples prior to sequencing. Using malaria clinical samples with over 80% human host DNA contamination, we show that the enzymatic treatment enriches Plasmodium falciparum DNA up to ∼9-fold and generates high-quality, nonbiased sequence reads covering >98% of 86,158 catalogued typeable single-nucleotide polymorphism loci.
Malaria parasites actively remodel the infected red blood cell (irbc) by exporting proteins into the host cell cytoplasm. The human parasite Plasmodium falciparum exports particularly large numbers of proteins, including proteins that establish a vesicular network allowing the trafficking of proteins onto the surface of irbcs that are responsible for tissue sequestration. Like P. falciparum, the rodent parasite P. berghei ANKA sequesters via irbc interactions with the host receptor CD36. We have applied proteomic, genomic, and reverse-genetic approaches to identify P. berghei proteins potentially involved in the transport of proteins to the irbc surface. A comparative proteomics analysis of P. berghei non-sequestering and sequestering parasites was used to determine changes in the irbc membrane associated with sequestration. Subsequent tagging experiments identified 13 proteins (Plasmodium export element (PEXEL)-positive as well as PEXEL-negative) that are exported into the irbc cytoplasm and have distinct localization patterns: a dispersed and/or patchy distribution, a punctate vesicle-like pattern in the cytoplasm, or a distinct location at the irbc membrane. Members of the PEXEL-negative BIR and PEXEL-positive Pb-fam-3 show a dispersed localization in the irbc cytoplasm, but not at the irbc surface. Two of the identified exported proteins are transported to the irbc membrane and were named erythrocyte membrane associated proteins. EMAP1 is a member of the PEXEL-negative Pb-fam-1 family, and EMAP2 is a PEXEL-positive protein encoded by a single copy gene; neither protein plays a direct role in sequestration. Our observations clearly indicate that P. berghei traffics a diverse range of proteins to different cellular locations via mechanisms that are analogous to those employed by P. falciparum. This information can be exploited to generate transgenic humanized rodent P. berghei parasites expressing chimeric P. berghei/P. falciparum proteins on the surface of rodent irbc, thereby opening new avenues for in vivo screening adjunct therapies that block sequestration.
Perceptual learning is the ability to improve perception through practice. Perceptual learning is usually specific for the task and features learned. For example, improvements in performance for a certain stimulus do not transfer if the stimulus is rotated by 90° or is presented at a different location. These findings are usually taken as evidence that orientation-specific, retinotopic encoding processes are changed during training. In this study, we used a novel masking paradigm in which the offset in an invisible, oblique vernier stimulus was perceived in an aligned vertical or horizontal flanking stimulus presented at a different location. Our results show that learning is specific for the perceived orientation of the vernier offset but not for its actual orientation and location. Specific encoding processes cannot be invoked to explain this improvement. We propose that perceptual learning involves changes in nonretinotopic, attentional readout processes.
perceptual learning; orientation specificity; vernier acuity; attention; consciousness
In malaria parasites the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (AT) rich Plasmodium DNA in E. coli. We overcome these roadblocks by demonstrating that a high integrity library of P. berghei genomic DNA (>77% AT) in a bacteriophage N15-based vector can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating Plasmodium berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to 10-fold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy.
Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform.
All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.
Next-generation sequencing; Ion torrent; Illumina; Pacific biosciences; MiSeq; PGM; SMRT; Bias; Genome coverage; GC-rich; AT-rich
To investigate the integration of features, we have developed a paradigm in which an element is rendered invisible by visual masking. Still, the features of the element are visible as part of other display elements presented at different locations and times (sequential metacontrast). In this sense, we can “transport” features non-retinotopically across space and time. The features of the invisible element integrate with features of other elements if and only if the elements belong to the same spatio-temporal group. The mechanisms of this kind of feature integration seem to be quite different from classical mechanisms proposed for feature binding. We propose that feature processing, binding, and integration occur concurrently during processes that group elements into wholes.
feature binding; feature processing; feature integration; sequential metacontrast paradigm; feature inheritance
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis, and the strains used worldwide represent a family of daughter strains with distinct genotypic characteristics. Here we report the complete genome sequence of M. bovis BCG Moreau, the strain in continuous use in Brazil for vaccine production since the 1920s.
The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cirs and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s) of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required.
The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages.
In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub-family A and B protein functions, including their contribution to antigenic variation and immune evasion.
The perception of a visual target can be strongly influenced by flanking stimuli. In static displays, performance on the target improves when the distance to the flanking elements increases- proposedly because feature pooling and integration vanishes with distance. Here, we studied feature integration with dynamic stimuli. We show that features of single elements presented within a continuous motion stream are integrated largely independent of spatial distance (and orientation). Hence, space based models of feature integration cannot be extended to dynamic stimuli. We suggest that feature integration is guided by perceptual grouping operations that maintain the identity of perceptual objects over space and time.
non-retinotopic processing; grouping; apparent motion; metacontrast masking; oblique effect; contrast polarity; attention; object file
So-called next-generation sequencing (NGS) has provided the ability to sequence on a massive scale at low cost, enabling biologists to perform powerful experiments and gain insight into biological processes. BamView has been developed to visualize and analyse sequence reads from NGS platforms, which have been aligned to a reference sequence. It is a desktop application for browsing the aligned or mapped reads [Ruffalo, M, LaFramboise, T, Koyutürk, M. Comparative analysis of algorithms for next-generation sequencing read alignment. Bioinformatics 2011;27:2790–6] at different levels of magnification, from nucleotide level, where the base qualities can be seen, to genome or chromosome level where overall coverage is shown. To enable in-depth investigation of NGS data, various views are provided that can be configured to highlight interesting aspects of the data. Multiple read alignment files can be overlaid to compare results from different experiments, and filters can be applied to facilitate the interpretation of the aligned reads. As well as being a standalone application it can be used as an integrated part of the Artemis genome browser, BamView allows the user to study NGS data in the context of the sequence and annotation of the reference genome. Single nucleotide polymorphism (SNP) density and candidate SNP sites can be highlighted and investigated, and read-pair information can be used to discover large structural insertions and deletions. The application will also calculate simple analyses of the read mapping, including reporting the read counts and reads per kilobase per million mapped reads (RPKM) for genes selected by the user.
Availability: BamView and Artemis are freely available software. These can be downloaded from their home pages:
Requirements: Java 1.6 or higher.
genome browser; next-generation sequencing; visualization; Artemis; BamView
Schistosomiasis is one of the most prevalent parasitic diseases, affecting millions of people in developing countries. Amongst the human-infective species, Schistosoma mansoni is also the most commonly used in the laboratory and here we present the systematic improvement of its draft genome. We used Sanger capillary and deep-coverage Illumina sequencing from clonal worms to upgrade the highly fragmented draft 380 Mb genome to one with only 885 scaffolds and more than 81% of the bases organised into chromosomes. We have also used transcriptome sequencing (RNA-seq) from four time points in the parasite's life cycle to refine gene predictions and profile their expression. More than 45% of predicted genes have been extensively modified and the total number has been reduced from 11,807 to 10,852. Using the new version of the genome, we identified trans-splicing events occurring in at least 11% of genes and identified clear cases where it is used to resolve polycistronic transcripts. We have produced a high-resolution map of temporal changes in expression for 9,535 genes, covering an unprecedented dynamic range for this organism. All of these data have been consolidated into a searchable format within the GeneDB (www.genedb.org) and SchistoDB (www.schistodb.net) databases. With further transcriptional profiling and genome sequencing increasingly accessible, the upgraded genome will form a fundamental dataset to underpin further advances in schistosome research.
Schistosomiasis is a disease caused by parasitic blood flukes of the genus Schistosoma. Human-infective species are prevalent in developing countries, where they represent a major disease burden as well as an impediment to socioeconomic development. In addition to its clinical relevance, Schistosoma mansoni is the species most widely used for laboratory experimentation. In 2009, the first draft of the S. mansoni and S. japonicum genomes were published. Both genome sequences represented a great step forward for schistosome research, but their highly fragmented nature compromised the quality of potential downstream analyses. In this study, we have substantially improved both the genome and the transcriptome resources for S. mansoni. We collated existing data and added deep DNA sequence data from clonal worms and RNA sequence data from four key time points in the life cycle of the parasite. We were able to identify transcribed regions to single-base resolution and have profiled gene expression from the free-living larvae to the early human parasitic stage. We uncovered extensive use of single transcripts from multiple genes, which the organism subsequently resolves by trans-splicing. All data from this study comprise a major new release of the genome, which is publicly and easily accessible.
Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.
We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.
We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.
Next-Generation Sequencing; Illumina; Library; Plasmodium falciparum; AT-rich; Malaria; Clinical isolate; PCR; Tetramethyammonium chloride; PCR-free; Isothermal; Linear; Exponential
Features of moving objects are non-retinotopically integrated along their motion trajectories as demonstrated by a variety of recent studies. The mechanisms of non-retinotopic feature integration are largely unknown. Here, we investigated the role of attention in non-retinotopic feature integration by using the sequential metacontrast paradigm. A central line was offset either to the left or right. A sequence of flanking lines followed eliciting the percept of two diverging motion streams. Although the central line was invisible, its offset was perceived within the streams. Observers attended to one stream. If an offset was introduced to one of the flanking lines in the attended stream, this offset integrated with the central line offset. No integration occurred when the offset was in the non-attended stream. Here, we manipulated the allocation of attention by using an auditory cueing paradigm. First, we show that mandatory non-retinotopic integration occurred even when the cue came long after the motion sequence. Second, we used more than two streams of which two could merge. Offsets in different streams were integrated when the streams merged. However, offsets of one stream were not integrated when this stream had to be ignored. We propose a hierarchical two stage model, in which motion grouping determines mandatory feature integration while attention selects motion streams for optional feature integration.
feature integration; non-retinotopic processing; metacontrast masking; motion grouping; attention; object
Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite.
Bursaphelenchus xylophilus is an important plant pathogen, responsible for an epidemic of pine wilt disease in Asia and Europe. B. xylophilus has acquired the ability to parasitise plants independently from other economically important nematodes and has a complex life cycle that includes fungal feeding and a stage associated with an insect, as well as plant parasitism. We have sequenced the genome of B. xylophilus and used it as a resource to understand disease mechanisms and the biological basis of its complex ecology. The ability to break down cellulose, the major component of the plant cell wall, is a major problem for plant parasitic nematodes as few animals can produce the required enzymes (cellulases). Previous work has shown that other plant parasitic nematodes have acquired cellulases from bacteria but we show that all Bursaphelenchus cellulases were most likely acquired independently from fungi. We also describe a complex set of genes encoding enzymes that can break down proteins and other molecules, perhaps reflecting the range of organisms with which B. xylophilus interacts during its life cycle. The genome sequence of Bursaphelenchus represents an important step forward in understanding its biology, and will contribute to efforts to control the devastating disease it causes.
DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methylation and a core region surrounding the transcriptional start site as informative surrogate for promoter methylation. We find 17% of the 873 analyzed genes differentially methylated in their 5′-untranslated regions (5′-UTR) and about one third of the differentially methylated 5′-UTRs to be inversely correlated with transcription. While our study was controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.
Plasmodium yoelii YM asexual blood stage parasites express
multiple members of the py235 gene family, part of the
super-family of genes including those coding for Plasmodium
vivax reticulocyte binding proteins and Plasmodium
falciparum RH proteins. We previously identified a Py235
erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is
recognized by protective mAb 25.77. Proteins recognized by a second protective
mAb 25.37 have been identified by mass spectrometry and are encoded by two
genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the
phenotype. The expression of the members of the py235 family in
both the WT and gene deletion parasites was measured by quantitative RT-PCR and
RNA-Seq. py235ebp-1 expression was undetectable in the knockout
parasite, but transcription of other members of the family was essentially
unaffected. The knockout parasites continued to react with mAb 25.77; and the
25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534
products. The PY01185 product was also identified as erythrocyte binding. There
was no clear change in erythrocyte invasion profile suggesting that the PY01185
gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by
serving as an invasion ligand although the molecular details of its interaction
with erythrocytes have not been examined. The PY01365, PY01185, and
PY05995/PY03534 genes are part of a distinct subset of the py235 family. In
P. falciparum, the RH protein genes are under epigenetic
control and expression correlates with binding to distinct erythrocyte receptors
and specific invasion pathways, whereas in P. yoelii YM all the
genes are expressed and deletion of one does not result in upregulation of
another. We propose that simultaneous expression of multiple Py235 ligands
enables invasion of a wide range of host erythrocytes even in the presence of
antibodies to one or more of the proteins and that this functional redundancy at
the protein level gives the parasite phenotypic plasticity in the absence of
differences in gene expression.
Malaria parasites invade erythrocytes where they develop and multiply before
bursting out and invading fresh cells. There are sequential steps to invasion;
early in the process, specific parasite proteins bind to molecules on the
surface of the erythrocyte. Tight binding forms a junction between parasite and
host cell leading to the next steps in the invasion process. Several of these
parasite proteins, which establish contact with the host cell surface, are coded
by gene families. One family, first described in the rodent parasite
Plasmodium yoelii and found in all
Plasmodium spp, is often referred to as the reticulocyte
binding ligand family. In P. yoelii the proteins are called
Py235 and are coded by at least eleven genes. Previously, we identified one
family member which is the target of protective antibodies that prevent parasite
invasion. Here we have deleted the gene for this protein and examined the
consequences. Other members of the family take the place of the missing protein
but their genes are not up-regulated. The family provides the parasite with the
potential to recognize erythrocytes with different surface receptors and evade
the binding of protective antibodies through plasticity at the level of its
Second-generation sequencing technologies have made large-scale sequencing projects commonplace. However, making use of these datasets often requires gene function to be ascribed genome wide. Although tool development has kept pace with the changes in sequence production, for tasks such as mapping, de novo assembly or visualization, genome annotation remains a challenge. We have developed a method to rapidly provide accurate annotation for new genomes using previously annotated genomes as a reference. The method, implemented in a tool called RATT (Rapid Annotation Transfer Tool), transfers annotations from a high-quality reference to a new genome on the basis of conserved synteny. We demonstrate that a Mycobacterium tuberculosis genome or a single 2.5 Mb chromosome from a malaria parasite can be annotated in less than five minutes with only modest computational resources. RATT is available at http://ratt.sourceforge.net.
Trypanosoma cruzi is the etiological agent of Chagas' disease, an endemic infection that causes thousands of deaths every year in Latin America. Therapeutic options remain inefficient, demanding the search for new drugs and/or new molecular targets. Such efforts can focus on proteins that are specific to the parasite, but analogous enzymes and enzymes with a three-dimensional (3D) structure sufficiently different from the corresponding host proteins may represent equally interesting targets. In order to find these targets we used the workflows MHOLline and AnEnΠ obtaining 3D models from homologous, analogous and specific proteins of Trypanosoma cruzi versus Homo sapiens.
We applied genome wide comparative modelling techniques to obtain 3D models for 3,286 predicted proteins of T. cruzi. In combination with comparative genome analysis to Homo sapiens, we were able to identify a subset of 397 enzyme sequences, of which 356 are homologous, 3 analogous and 38 specific to the parasite.
In this work, we present a set of 397 enzyme models of T. cruzi that can constitute potential structure-based drug targets to be investigated for the development of new strategies to fight Chagas' disease. The strategies presented here support the concept of structural analysis in conjunction with protein functional analysis as an interesting computational methodology to detect potential targets for structure-based rational drug design. For example, 2,4-dienoyl-CoA reductase (EC 18.104.22.168) and triacylglycerol lipase (EC 22.214.171.124), classified as analogous proteins in relation to H. sapiens enzymes, were identified as new potential molecular targets.
Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.
A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.
This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.