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

Nematode.net update 2011: addition of data sets and tools featuring next-generation sequencing data

Nematode.net (http://nematode.net) has been a publicly available resource for studying nematodes for over a decade. In the past 3 years, we reorganized Nematode.net to provide more user-friendly navigation through the site, a necessity due to the explosion of data from next-generation sequencing platforms. Organism-centric portals containing dynamically generated data are available for over 56 different nematode species. Next-generation data has been added to the various data-mining portals hosted, including NemaBLAST and NemaBrowse. The NemaPath metabolic pathway viewer builds associations using KOs, rather than ECs to provide more accurate and fine-grained descriptions of proteins. Two new features for data analysis and comparative genomics have been added to the site. NemaSNP enables the user to perform population genetics studies in various nematode populations using next-generation sequencing data. HelmCoP (Helminth Control and Prevention) as an independent component of Nematode.net provides an integrated resource for storage, annotation and comparative genomics of helminth genomes to aid in learning more about nematode genomes, as well as drug, pesticide, vaccine and drug target discovery. With this update, Nematode.net will continue to realize its original goal to disseminate diverse bioinformatic data sets and provide analysis tools to the broad scientific community in a useful and user-friendly manner

The prevalence of species and strains in the human microbiome: A resource for experimental efforts

Experimental efforts to characterize the human microbiota often use bacterial strains that were chosen for historical rather than biological reasons. Here, we report an analysis of 380 whole-genome shotgun samples from 100 subjects from the NIH Human Microbiome Project. By mapping their reads to 1,751 reference genome sequences and analyzing the resulting relative strain abundance in each sample we present metrics and visualizations that can help identify strains of interest for experimentalists. We also show that approximately 14 strains of 10 species account for 80% of the mapped reads from a typical stool sample, indicating that the function of a community may not be irreducibly complex. Some of these strains account for >20% of the sequence reads in a subset of samples but are absent in others, a dichotomy that could underlie biological differences among subjects. These data should serve as an important strain selection resource for the community of researchers who take experimental approaches to studying the human microbiota

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

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

Nematode.net update 2008: improvements enabling more efficient data mining and comparative nematode genomics

Nematode.net (http://nematode.net) is a publicly available resource dedicated to the study of parasitic nematodes. In 2000, the Genome Center at Washington University (GC) joined a consortium including the Nematode Genomics group in Edinburgh, and the Pathogen Sequencing Unit of the Sanger Institute to generate expressed sequence tags (ESTs) as an inexpensive and efficient solution for gene discovery in parasitic nematodes. As of 2008 the GC, sampling key parasites of humans, animals and plants, has generated over 500 000 ESTs and 1.2 million genome survey sequences from more than 30 non-Caenorhabditis elegans nematodes. Nematode.net was implemented to offer user-friendly access to data produced by this project. In addition to sequence data, the site hosts: assembled NemaGene clusters in GBrowse views characterizing composition and protein homology, functional Gene Ontology annotations presented via the AmiGO browser, KEGG-based graphical display of NemaGene clusters mapped to metabolic pathways, codon usage tables, NemFam protein families which represent conserved nematode-restricted coding sequences not found in public protein databases, a web-based WU-BLAST search tool that allows complex querying and other assorted resources. The primary aim of Nematode.net is the dissemination of this diverse collection of information to the broader scientific community in a way that is useful, consistent, centralized and enduring

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

Mining Novel Effector Proteins from the Esophageal Gland Cells of Meloidogyne incognita

Meloidogyne incognita is one of the most economically damaging plant pathogens in agriculture and horticulture. Identifying and characterizing the effector proteins which M. incognita secretes into its host plants during infection is an important step toward finding new ways to manage this pest. In this study, we have identified the cDNAs for 18 putative effectors (i.e., proteins that have the potential to facilitate M. incognita parasitism of host plants). These putative effectors are secretory proteins that do not contain transmembrane domains and whose genes are specifically expressed in the secretory gland cells of the nematode, indicating that they are likely secreted from the nematode through its stylet. We have determined that, in the plant cells, these putative effectors are likely to localize to the cytoplasm. Furthermore, the transcripts of many of these novel effectors are specifically upregulated during different stages of the nematode\u27s life cycle, indicating that they function at specific stages during M. incognita parasitism. The predicted proteins showed little to no homology to known proteins from free-living nematode species, suggesting that they evolved recently to support the parasitic lifestyle. On the other hand, several of the effectors are part of gene families within the M. incognita genome as well as that of M. hapla, which points to an important role that these putative effectors are playing in both parasites. With the discovery of these putative effectors, we have increased our knowledge of the effector repertoire utilized by root-knot nematodes to infect, feed on, and reproduce on their host plants. Future studies investigating the roles that these proteins play in planta will help mitigate the effects of this damaging pest

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