Identification and characterization of alternative splicing in parasitic nematode transcriptomes

BACKGROUND: Alternative splicing (AS) of mRNA is a vital mechanism for enhancing genomic complexity in eukaryotes. Spliced isoforms of the same gene can have diverse molecular and biological functions and are often differentially expressed across various tissues, times, and conditions. Thus, AS has important implications in the study of parasitic nematodes with complex life cycles. Transcriptomic datasets are available from many species, but data must be revisited with splice-aware assembly protocols to facilitate the study of AS in helminthes. METHODS: We sequenced cDNA from the model worm Caenorhabditis elegans using 454/Roche technology for use as an experimental dataset. Reads were assembled with Newbler software, invoking the cDNA option. Several combinations of parameters were tested and assembled transcripts were verified by comparison with previously reported C. elegans genes and transcript isoforms and with Illumina RNAseq data. RESULTS: Thoughtful adjustment of program parameters increased the percentage of assembled transcripts that matched known C. elegans sequences, decreased mis-assembly rates (i.e., cis- and trans-chimeras), and improved the coverage of the geneset. The optimized protocol was used to update de novo transcriptome assemblies from nine parasitic nematode species, including important pathogens of humans and domestic animals. Our assemblies indicated AS rates in the range of 20-30%, typically with 2-3 transcripts per AS locus, depending on the species. Transcript isoforms from the nine species were translated and searched for similarity to known proteins and functional domains. Some 21 InterPro domains, including several involved in nucleotide and chromatin binding, were statistically correlated with AS genetic loci. In most cases, the Roche/454 data explored in this study are the only sequences available from the species in question; however, the recently published genome of the human hookworm Necator americanus provided an additional opportunity to validate our results. CONCLUSIONS: Our optimized assembly parameters facilitated the first survey of AS among parasitic nematodes. The nine transcriptome assemblies, their protein translations, and basic annotations are available from Nematode.net as a resource for the research community. These should be useful for studies of specific genes and gene families of interest as well as for curating draft genome assemblies as they become available

Characterizing Ancylostoma caninum transcriptome and exploring nematode parasitic adaptation

<p>Abstract</p> <p>Background</p> <p>Hookworm infection is one of the most important neglected diseases in developing countries, with approximately 1 billion people infected worldwide. To better understand hookworm biology and nematode parasitism, the present study generated a near complete transcriptome of the canine hookworm <it>Ancylostoma caninum </it>to a very high coverage using high throughput technology, and compared it to those of the free-living nematode <it>Caenorhabditis elegans </it>and the parasite <it>Brugia malayi</it>.</p> <p>Results</p> <p>The generated transcripts from four developmental stages, infective L3, serum stimulated L3, adult male and adult female, covered 93% of the <it>A. caninum </it>transcriptome. The broad diversity among nematode transcriptomes was confirmed, and an impact of parasitic adaptation on transcriptome diversity was inferred. Intra-population analysis showed that <it>A. caninum </it>has higher coding sequence diversity than humans. Examining the developmental expression profiles of <it>A. caninum </it>revealed major transitions in gene expression from larval stages to adult. Adult males expressed the highest number of selectively expressed genes, but adult female expressed the highest number of selective parasitism-related genes. Genes related to parasitism adaptation and <it>A. caninum </it>specific genes exhibited more expression selectivity while those conserved in nematodes tend to be consistently expressed. Parasitism related genes were expressed more selectively in adult male and female worms. The comprehensive analysis of digital expression profiles along with transcriptome comparisons enabled identification of a set of parasitism genes encoding secretory proteins in animal parasitic nematode.</p> <p>Conclusions</p> <p>This study validated the usage of deep sequencing for gene expression profiling. Parasitic adaptation of the canine hookworm is related to its diversity and developmental dynamics. This comprehensive comparative genomic and expression study substantially improves our understanding of the basic biology and parasitism of hookworms and, is expected, in the long run, to accelerate research toward development of vaccines and novel anthelmintics.</p

Using existing drugs as leads for broad spectrum anthelmintics targeting protein kinases

As one of the largest protein families, protein kinases (PKs) regulate nearly all processes within the cell and are considered important drug targets. Much research has been conducted on inhibitors for PKs, leading to a wealth of compounds that target PKs that have potential to be lead anthelmintic drugs. Identifying compounds that have already been developed to treat neglected tropical diseases is an attractive way to obtain lead compounds inexpensively that can be developed into much needed drugs, especially for use in developing countries. In this study, PKs from nematodes, hosts, and DrugBank were identified and classified into kinase families and subfamilies. Nematode proteins were placed into orthologous groups that span the phylum Nematoda. A minimal kinome for the phylum Nematoda was identified, and properties of the minimal kinome were explored. Orthologous groups from the minimal kinome were prioritized for experimental testing based on RNAi phenotype of the Caenorhabditis elegans ortholog, transcript expression over the life-cycle and anatomic expression patterns. Compounds linked to targets in DrugBank belonging to the same kinase families and subfamilies in the minimal nematode kinome were extracted. Thirty-five compounds were tested in the non-parasitic C. elegans and active compounds progressed to testing against nematode species with different modes of parasitism, the blood-feeding Haemonchus contortus and the filarial Brugia malayi. Eighteen compounds showed efficacy in C. elegans, and six compounds also showed efficacy in at least one of the parasitic species. Hypotheses regarding the pathway the compounds may target and their molecular mechanism for activity are discussed

Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

<p>Abstract</p> <p>Background</p> <p>Most filarial nematodes contain <it>Wolbachia </it>symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in <it>Wolbachia </it>and adult female <it>Brugia malayi</it>.</p> <p>Methods</p> <p><it>Brugia malayi </it>infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared <it>Wolbachia </it>and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.</p> <p>Results and discussion</p> <p>Results showed significant changes in expression for 200 <it>Wolbachia </it>(29% of <it>Wolbachia </it>genes with expression signals in untreated worms) and 546 <it>B. malayi </it>array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed <it>Wolbachia </it>genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on <it>B. malayi </it>gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).</p> <p>Conclusions</p> <p>Doxycycline reduced <it>Wolbachia </it>and significantly decreased bacterial gene expression. <it>Wolbachia </it>ribosomes are believed to be the primary biological target for doxycycline in filarial worms. <it>B. malayi </it>genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many <it>B. malayi </it>genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of <it>Wolbachia </it>so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between <it>Wolbachia </it>and <it>B. malayi</it>.</p

NemaPath: online exploration of KEGG-based metabolic pathways for nematodes

<p>Abstract</p> <p>Background</p> <p>Nematode.net <url>http://www.nematode.net</url> is a web-accessible resource for investigating gene sequences from parasitic and free-living nematode genomes. Beyond the well-characterized model nematode <it>C. elegans</it>, over 500,000 expressed sequence tags (ESTs) and nearly 600,000 genome survey sequences (GSSs) have been generated from 36 nematode species as part of the Parasitic Nematode Genomics Program undertaken by the Genome Center at Washington University School of Medicine. However, these sequencing data are not present in most publicly available protein databases, which only include sequences in Swiss-Prot. Swiss-Prot, in turn, relies on GenBank/Embl/DDJP for predicted proteins from complete genomes or full-length proteins.</p> <p>Description</p> <p>Here we present the NemaPath pathway server, a web-based pathway-level visualization tool for navigating putative metabolic pathways for over 30 nematode species, including 27 parasites. The NemaPath approach consists of two parts: 1) a backend tool to align and evaluate nematode genomic sequences (curated EST contigs) against the annotated Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database; 2) a web viewing application that displays annotated KEGG pathway maps based on desired confidence levels of primary sequence similarity as defined by a user. NemaPath also provides cross-referenced access to nematode genome information provided by other tools available on Nematode.net, including: detailed NemaGene EST cluster information; putative translations; GBrowse EST cluster views; links from nematode data to external databases for corresponding synonymous <it>C. elegans </it>counterparts, subject matches in KEGG's gene database, and also KEGG Ontology (KO) identification.</p> <p>Conclusion</p> <p>The NemaPath server hosts metabolic pathway mappings for 30 nematode species and is available on the World Wide Web at <url>http://nematode.net/cgi-bin/keggview.cgi</url>. The nematode source sequences used for the metabolic pathway mappings are available via FTP <url>http://www.nematode.net/FTP/index.php</url>, as provided by the Genome Center at Washington University School of Medicine.</p

Molecular determinants archetypical to the phylum Nematoda

<p>Abstract</p> <p>Background</p> <p>Nematoda diverged from other animals between 600–1,200 million years ago and has become one of the most diverse animal phyla on earth. Most nematodes are free-living animals, but many are parasites of plants and animals including humans, posing major ecological and economical challenges around the world.</p> <p>Results</p> <p>We investigated phylum-specific molecular characteristics in Nematoda by exploring over 214,000 polypeptides from 32 nematode species including 27 parasites. Over 50,000 nematode protein families were identified based on primary sequence, including ~10% with members from at least three different species. Nearly 1,600 of the multi-species families did not share homology to Pfam domains, including a total of 758 restricted to Nematoda. Majority of the 462 families that were conserved among both free-living and parasitic species contained members from multiple nematode clades, yet ~90% of the 296 parasite-specific families originated only from a single clade. Features of these protein families were revealed through extrapolation of essential functions from observed RNAi phenotypes in <it>C. elegans</it>, bioinformatics-based functional annotations, identification of distant homology based on protein folds, and prediction of expression at accessible nematode surfaces. In addition, we identified a group of nematode-restricted sequence features in energy-generating electron transfer complexes as potential targets for new chemicals with minimal or no toxicity to the host.</p> <p>Conclusion</p> <p>This study identified and characterized the molecular determinants that help in defining the phylum Nematoda, and therefore improved our understanding of nematode protein evolution and provided novel insights for the development of next generation parasite control strategies.</p

Discovery of anthelmintic drug targets and drugs using chokepoints in nematode metabolic pathways

Parasitic roundworm infections plague more than 2 billion people (1/3 of humanity) and cause drastic losses in crops and livestock. New anthelmintic drugs are urgently needed as new drug resistance and environmental concerns arise. A "chokepoint reaction" is defined as a reaction that either consumes a unique substrate or produces a unique product. A chokepoint analysis provides a systematic method of identifying novel potential drug targets. Chokepoint enzymes were identified in the genomes of 10 nematode species, and the intersection and union of all chokepoint enzymes were found. By studying and experimentally testing available compounds known to target proteins orthologous to nematode chokepoint proteins in public databases, this study uncovers features of chokepoints that make them successful drug targets. Chemogenomic screening was performed on drug-like compounds from public drug databases to find existing compounds that target homologs of nematode chokepoints. The compounds were prioritized based on chemical properties frequently found in successful drugs and were experimentally tested using Caenorhabditis elegans. Several drugs that are already known anthelmintic drugs and novel candidate targets were identified. Seven of the compounds were tested in Caenorhabditis elegans and three yielded a detrimental phenotype. One of these three drug-like compounds, Perhexiline, also yielded a deleterious effect in Haemonchus contortus and Onchocerca lienalis, two nematodes with divergent forms of parasitism. Perhexiline, known to affect the fatty acid oxidation pathway in mammals, caused a reduction in oxygen consumption rates in C. elegans and genome-wide gene expression profiles provided an additional confirmation of its mode of action. Computational modeling of Perhexiline and its target provided structural insights regarding its binding mode and specificity. Our lists of prioritized drug targets and drug-like compounds have potential to expedite the discovery of new anthelmintic drugs with broad-spectrum efficacy

Genome of the human hookworm \u3ci\u3eNecator americanus\u3c/i\u3e

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm\u27s invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases