Gary P. Dillon

The genome of the blood fluke Schistosoma mansoni

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni

Schistosoma mansoni is the primary causative agent of schistosomiasis, which affects 200 million individuals in 74 countries. We generated 163,000 expressed-sequence tags (ESTs) from normalized cDNA libraries from six selected developmental stages of the parasite, resulting in 31,000 assembled sequences and 92% sampling of an estimated 14,000 gene complement. By analyzing automated Gene Ontology assignments, we provide a detailed view of important S. mansoni biological systems, including characterization of metazoa-specific and eukarya-conserved genes. Phylogenetic analysis suggests an early divergence from other metazoa. The data set provides insights into the molecular mechanisms of tissue organization, development, signaling, sexual dimorphism, host interactions and immune evasion and identifies novel proteins to be investigated as vaccine candidates and potential drug targets.

A systematically improved high quality genome and transcriptome of the human blood fluke Schistosoma mansoni.

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 parasites 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.

RATT: Rapid Annotation Transfer Tool

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.

Protein variation in blood-dwelling schistosome worms generated by differential splicing of micro-exon gene transcripts

Schistosoma mansoni is a well-adapted blood-dwelling parasitic helminth, persisting for decades in its human host despite being continually exposed to potential immune attack. Here, we describe in detail micro-exon genes (MEG) in S. mansoni, some present in multiple copies, which represent a novel molecular system for creating protein variation through the alternate splicing of short (< or =36 bp) symmetric exons organized in tandem. Analysis of three closely related copies of one MEG family allowed us to trace several evolutionary events and propose a mechanism for micro-exon generation and diversification. Microarray experiments show that the majority of MEGs are up-regulated in life cycle stages associated with establishment in the mammalian host after skin penetration. Sequencing of RT-PCR products allowed the description of several alternate splice forms of micro-exon genes, highlighting the potential use of these transcripts to generate a complex pool of protein variants. We obtained direct evidence for the existence of such pools by proteomic analysis of secretions from migrating schistosomula and mature eggs. Whole-mount in situ hybridization and immunolocalization showed that MEG transcripts and proteins were restricted to glands or epithelia exposed to the external environment. The ability of schistosomes to produce a complex pool of variant proteins aligns them with the other major groups of blood parasites, but using a completely different mechanism. We believe that our data open a new chapter in the study of immune evasion by schistosomes, and their ability to generate variant proteins could represent a significant obstacle to vaccine development.

Gene Expression Patterns in Larval Schistosoma mansoni Associated with Infection of the Mammalian Host

Background The infective schistosome cercaria develops within the intramolluscan daughter sporocyst from an undifferentiated germ ball, during which synthesis of proteins essential for infection occurs. When the aquatic cercaria locates the mammalian host it rapidly penetrates into the epidermis using glandular secretions. It then undergoes metamorphosis into the schistosomulum, including replacement of its tegument surface membranes, a process taking several days before it exits the skin. Patterns of gene expression underlying this transition have been characterised. Methods and Principal Findings All gene models from the S. mansoni genome (www.GeneDB.org) were incorporated into a high-density oligonucleotide array. Double-stranded cDNA from germ balls, cercariae, and day 3 schistosomula was hybridised to the array without amplification. Statistical analysis was performed using Bioconductor to reveal differentially transcribed loci. Genes were categorised on the basis of biological process, tissue association or molecular function to aid understanding of the complex processes occurring. Genes necessary for DNA replication were enriched only in the germ ball, while those involved in translation were up-regulated in the germ ball and/or day 3 schistosomulum. Different sets of developmental genes were up-regulated at each stage. A large number of genes encoding elastases and invadolysins, and some venom allergen-like proteins were up-regulated in the germ ball, those encoding cysteine and aspartic proteases in the cercaria and schistosomulum. Micro exon genes encoding variant secreted proteins were highly up-regulated in the schistosomulum along with tegument and gut-associated genes, coincident with remodelling of the parasite body. Genes encoding membrane proteins were prominently up-regulated in the cercaria and/or day 3 schistosomulum. Conclusions/Significance Our study highlights an expanded number of transcripts encoding proteins potentially involved in skin invasion. It illuminates the process of metamorphosis into the schistosomulum and highlights the very early activation of gut-associated genes whilst revealing little change in the parasites energy metabolism or stress responses.

Elimination of Schistosoma mansoni Adult Worms by Rhesus Macaques: Basis for a Therapeutic Vaccine?

BACKGROUND Among animal models of schistosomiasis, the rhesus macaque is unique in that an infection establishes but egg excretion rapidly diminishes, potentially due to loss of adult worms from the portal system via shunts or death by immune attack. PRINCIPAL FINDINGS To investigate this, six rhesus macaques were exposed to Schistosoma mansoni cercariae and the infection monitored until portal perfusion at 18 weeks. Despite a wide variation in worm numbers recovered, fecal egg output and circulating antigen levels indicated that a substantial population had established in all animals. Half the macaques had portal hypertension but only one had portacaval shunts, ruling out translocation to the lungs as the reason for loss of adult burden. Many worms had a shrunken and pallid appearance, with degenerative changes in intestines and reproductive organs. Tegument, gut epithelia and muscles appeared cytologically intact but the parenchyma was virtually devoid of content. An early and intense IgG production correlated with low worm burden at perfusion, and blood-feeding worms cultured in the presence of serum from these animals had stunted growth. Using immunoproteomics, gut digestive enzymes, tegument surface hydrolases and antioxidant enzymes were identified as targets of IgG in the high responder animals. SIGNIFICANCE It appears that worms starve to death after cessation of blood feeding, as a result of antibody-mediated processes. We suggest that proteins in the three categories above, formulated to trigger the appropriate mechanisms operating in rhesus macaques, would have both prophylactic and therapeutic potential as a human vaccine.

Altered patterns of gene expression underlying the enhanced immunogenicity of radiation-attenuated schistosomes.

Background Schistosome cercariae only elicit high levels of protective immunity against a challenge infection if they are optimally attenuated by exposure to ionising radiation that truncates their migration in the lungs. However, the underlying molecular mechanisms responsible for the altered phenotype of the irradiated parasite that primes for protection have yet to be identified. Methodology/Principal Findings We have used a custom microarray comprising probes derived from lung-stage parasites to compare patterns of gene expression in schistosomula derived from normal and irradiated cercariae. These were transformed in vitro and cultured for four, seven, and ten days to correspond in development to the priming parasites, before RNA extraction. At these late times after the radiation insult, transcript suppression was the principal feature of the irradiated larvae. Individual gene analysis revealed that only seven were significantly down-regulated in the irradiated versus normal larvae at the three time-points; notably, four of the protein products are present in the tegument or associated with its membranes, perhaps indicating a perturbed function. Grouping of transcripts using Gene Ontology (GO) and subsequent Gene Set Enrichment Analysis (GSEA) proved more informative in teasing out subtle differences. Deficiencies in signalling pathways involving G-protein–coupled receptors suggest the parasite is less able to sense its environment. Reduction of cytoskeleton transcripts could indicate compromised structure which, coupled with a paucity of neuroreceptor transcripts, may mean the parasite is also unable to respond correctly to external stimuli. Conclusions/Significance The transcriptional differences observed are concordant with the known extended transit of attenuated parasites through skin-draining lymph nodes and the lungs: prolonged priming of the immune system by the parasite, rather than over-expression of novel antigens, could thus explain the efficacy of the irradiated vaccine.