Assessment and improvement of the Plasmodium yoelii yoelii genome annotation through comparative analysis

Motivation: The sequencing of the Plasmodium yoelii genome, a model rodent malaria parasite, has greatly facilitated research for the development of new drug and vaccine candidates against malaria. Unfortunately, only preliminary gene models were annotated on the partially sequenced genome, mostly by in silico gene prediction, and there has been no major improvement of the annotation since 2002

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

A novel series of compositionally biased substitution matrices for comparing Plasmodium proteins

<p>Abstract</p> <p>Background</p> <p>The most common substitution matrices currently used (BLOSUM and PAM) are based on protein sequences with average amino acid distributions, thus they do not represent a fully accurate substitution model for proteins characterized by a biased amino acid composition. This problem has been addressed recently by adjusting existing matrices, however, to date, no empirical approach has been taken to build matrices which offer a substitution model for comparing proteins sharing an amino acid compositional bias. Here, we present a novel procedure to construct series of symmetrical substitution matrices to align proteins from similarly biased <it>Plasmodium </it>proteomes.</p> <p>Results</p> <p>We generated substitution matrices by selecting from the BLOCKS database those multiple alignments with a compositional bias similar to that of <it>P. falciparum </it>and <it>P. yoelii </it>proteins. A novel 'fuzzy' clustering method was adopted to group sequences within these alignments, showing that this method retains more complete information on the amino acid substitutions when compared to hierarchical clustering. We assessed the performance against the BLOSUM62 series and showed that the usage of our matrices results in an improvement in the performance of BLAST database searches, greatly reducing the number of false positive hits. We then demonstrated applications of the use of novel matrices to improve the annotation of homologs between the two <it>Plasmodium </it>species and to classify members of the <it>P. falciparum </it>RIFIN/STEVOR family.</p> <p>Conclusion</p> <p>We confirmed that in the case of compositionally biased proteins, standard BLOSUM matrices are not suited for optimal alignments, and specific substitution matrices are required. In addition, we showed that the usage of these matrices leads to a reduction of false positive hits, facilitating the automatic annotation process.</p

Integration and mining of malaria molecular, functional and pharmacological data: how far are we from a chemogenomic knowledge space?

The organization and mining of malaria genomic and post-genomic data is highly motivated by the necessity to predict and characterize new biological targets and new drugs. Biological targets are sought in a biological space designed from the genomic data from Plasmodium falciparum, but using also the millions of genomic data from other species. Drug candidates are sought in a chemical space containing the millions of small molecules stored in public and private chemolibraries. Data management should therefore be as reliable and versatile as possible. In this context, we examined five aspects of the organization and mining of malaria genomic and post-genomic data: 1) the comparison of protein sequences including compositionally atypical malaria sequences, 2) the high throughput reconstruction of molecular phylogenies, 3) the representation of biological processes particularly metabolic pathways, 4) the versatile methods to integrate genomic data, biological representations and functional profiling obtained from X-omic experiments after drug treatments and 5) the determination and prediction of protein structures and their molecular docking with drug candidate structures. Progresses toward a grid-enabled chemogenomic knowledge space are discussed.Comment: 43 pages, 4 figures, to appear in Malaria Journa

Identification of two new protective pre-erythrocytic malaria vaccine antigen candidates

<p>Abstract</p> <p>Background</p> <p>Despite years of effort, a licensed malaria vaccine is not yet available. One of the obstacles facing the development of a malaria vaccine is the extensive heterogeneity of many of the current malaria vaccine antigens. To counteract this antigenic diversity, an effective malaria vaccine may need to elicit an immune response against multiple malaria antigens, thereby limiting the negative impact of variability in any one antigen. Since most of the malaria vaccine antigens that have been evaluated in people have not elicited a protective immune response, there is a need to identify additional protective antigens. In this study, the efficacy of three pre-erythrocytic stage malaria antigens was evaluated in a <it>Plasmodium yoelii</it>/mouse protection model.</p> <p>Methods</p> <p>Mice were immunized with plasmid DNA and vaccinia virus vectors that expressed one, two or all three <it>P. yoelii </it>vaccine antigens. The immunized mice were challenged with 300 <it>P. yoelii </it>sporozoites and evaluated for subsequent infection.</p> <p>Results</p> <p>Vaccines that expressed any one of the three antigens did not protect a high percentage of mice against a <it>P. yoelii </it>challenge. However, vaccines that expressed all three antigens protected a higher percentage of mice than a vaccine that expressed PyCSP, the most efficacious malaria vaccine antigen. Dissection of the multi-antigen vaccine indicated that protection was primarily associated with two of the three <it>P. yoelii </it>antigens. The protection elicited by a vaccine expressing these two antigens exceeded the sum of the protection elicited by the single antigen vaccines, suggesting a potential synergistic interaction.</p> <p>Conclusions</p> <p>This work identifies two promising malaria vaccine antigen candidates and suggests that a multi-antigen vaccine may be more efficacious than a single antigen vaccine.</p

Comparative Genome Analysis of Malaria Parasite Species

With over 200 million infections and up to one million deaths every year, malaria remains one of the most devastating infectious diseases affecting humans. Over the last few years, complete genome sequences of both human and non-human malaria parasite species have become available, adding comparative genomics to the toolbox of molecular biologists to study the genetic basis of human virulence. In this thesis, I computationally compared the published genomes of seven malaria parasite species with the aim to gain new insights into genes underlying human virulence. This comparison was performed using two complementary approaches. In the first approach, I used whole-genome synteny analysis to find genes present in human but not non-human malaria parasites. In the second approach, I first clustered virulence-associated genes into gene families and then examined these gene families for species-specific differences. Both comparisons resulted in interesting gene lists. Synteny analysis identified three key enzymes of the thiamine (vitamin B1) biosynthesis pathway to be present in human but not rodent malaria parasites, indicating that these two groups of parasites differ in their ability to synthesize vitamin B1 de novo. My gene family classification exposed within the largest and highly divergent surface antigen gene family pir a group of unusually well conserved orthologs, which should be considered as high-priority targets for experimental characterization and vaccine development. In conclusion, this thesis highlights genes and pathways that are different between human and non-human malaria parasites and therefore could play important roles in human virulence. Experimental studies can now be initiated to confirm virulence-associated functions and to explore their potential value for drug and vaccine development

Transcriptional regulation of yir genes in Plasmodium yoelii yoelii infected erythrocytes

In 1998, a large multi gene family was discovered in Plasmodium vivax (del Portillo et al., 1998). This multigene family was termed vir, and later studies showed that vir homologues existed in the three rodent malaria species, Plasmodium chabaudi (cir), Plasmodium berghei (bir) and Plasmodium yoelii (yir). By 5x coverage sequencing of Plasmodium yoelii 17X, 838 yir genes were predicted (Carlton et al., 2002), making this the largest known multi gene family in Plasmodium. YIR proteins are expressed at the surface of infected erythrocytes (Cunningham et al., 2005), and therefore this family is thought to be involved in antigenic variation. The aim of this thesis was to examine how P.yoelii regulates transcription of the yir family. The yir gene structure was verified experimentally, and phylogenetic analysis showed that yir genes could be divided into five supergroups consisting of yir genes with different sizes and subtelomeric localisation. In the blood stages, numerous yir genes were transcribed from all the supergroups in immunocompromised mice. However, a maximum of two yir genes were transcribed in single infected erythrocytes at the Schizont stage, which suggested strong silencing mechanisms. The transcriptional start and polyadenylation sites were identified experimentally, and it was found that both occurred at highly conserved motifs. In addition, the transcription initiation site was located close to an unusual and universally conserved triple-repeat motif, and it was found that all yir transcripts in two populations of parasites initiated downstream of this motif. Transfection experiments were performed in order to examine the role of this motif, but no solid conclusions could be drawn from these. Several alternative splicing events were detected in the yir 5'UTRs, and one of these led to exon 1 skipping of a yir gene. Through bioinformatic analysis of yir 5' intergenic regions, it was found that the UTR introns had a discrete distribution amongst the yir supergroups

Plasmodium yoelii acetyl-coa carboxylase : detection and characterisation of the recombinant biotinoyl domain.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.Human malaria, caused by four species of the intracellular protozoan parasite Plasmodium, is a major health and economic burden in the tropics where the disease is endemic. The biotindependent enzyme acetyl-CoA carboxylase catalyses the commitment step in de novo fatty acid biosynthesis in several organisms. Acetyl-CoA carboxylase is a target for anti-parasitic drug development due to its relevance in membrane biogenesis. This study describes the detection of acetyl-CoA carboxylase and the partial characterisation of the biotinoyl domain of the enzyme of the mouse malaria parasite, Plasmodium yoelii. Acetyl-CoA carboxylase mRNA was detected by RT-PCR performed on total RNA isolated from P. yoelii 17XL-infected mouse erythrocytes using primers designed from PY01695 ORF of the Plasmodb-published MALPY00458 gene of P. yoelii 17XNL. The RT-PCR was confirmed by sequencing and comparative analysis of the sequenced RT-PCR cDNA products. Northern blot analysis performed on total RNA using probes designed from a 1 kb region of the gene showed that the transcript was greater than the predicted 8.7 kb ORF. An immunogenic peptide corresponding to the P. yoelii theoretical acetyl-CoA carboxylase sequence was selected using epitope prediction and multiple sequence alignment algorithms. The immunogenic peptide was coupled to rabbit albumin carrier for immunisation in chickens and the affinity purified antibody titre was approximately 25 mg. The anti-peptide antibodies detected a 330 kD protein in P. yoelii lysate blot, which corresponds to the predicted size of the enzyme. The enzyme was also detected in situ by immunofluorescence microscopy using the anti-peptide antibodies. A 1 kb region of the P. yoelii acetyl-CoA carboxylase gene containing the biotinoyl domain was cloned and expressed in E. coli as 66 kD GST-tag and 45 kD His-tag protein. Both recombinant biotinoyl proteins were shown to contain bound biotin using peroxidaseconjugated avidin-biotin detection system. This suggested in vivo biotinylation of the recombinant P. yoelii biotinoyl protein, possibly by the E. coli biotin protein ligase. The Proscan™ and the NetPhos 2.0™ algorithms were used to predict protein kinase phosphorylation sites on the biotin carboxylase and the carboxyltransferase domains of the enzyme. The three-dimensional structure of the biotinoyl and the biotin carboxylase domains were predicted using the SWISS-MODEL™ homology modelling algorithm. Homology modelling revealed a similarity in the 3D conformation of the predicted P. yoelii biotinoyl domain and the E. coli biotinoyl protein with negligible root mean square deviation. The model also revealed the possibility of inhibiting P. yoelii and falciparum acetyl-CoA carboxylases with soraphen A based on the similarity in conformation with S. cerevisiae biotin carboxylase and the stereochemical properties of the residues predicted to interact with soraphen A. This study demonstrated that malaria parasite expresses acetyl-CoA carboxylase and, combined with data on other enzymes involved in fatty acid metabolism suggests that the parasite synthesizes fatty acids de novo. This enzyme could be a target for rational drug design