Evolutionary analysis of species-specific duplications in flatworm genomes.

Platyhelminthes, also known as flatworms, is a phylum of bilaterian invertebrates infamous for their parasitic representatives. The classes Cestoda, Monogenea, and Trematoda comprise parasitic helminths inhabiting multiple hosts, including fishes, humans, and livestock, and are responsible for considerable economic damage and burden on human health. As in other animals, the genomes of flatworms have a wide variety of paralogs, genes related via duplication, whose origins could be mapped throughout the evolution of the phylum. Through in-silico analysis, we studied inparalogs, i.e., species-specific duplications, focusing on their biological functions, expression changes, and evolutionary rate. These genes are thought to be key players in the adaptation process of species to each particular niche. Our results showed that genes related with specific functional terms, such as response to stress, transferase activity, oxidoreductase activity, and peptidases, are overrepresented among inparalogs. This trend is conserved among species from different classes, including free-living species. Available expression data from Schistosoma mansoni, a parasite from the trematode class, demonstrated high conservation of expression patterns between inparalogs, but with notable exceptions, which also display evidence of rapid evolution. We discuss how natural selection may operate to maintain these genes and the particular duplication models that fit better to the observations. Our work supports the critical role of gene duplication in the evolution of flatworms, representing the first study of inparalogs evolution at the genome-wide level in this group.

Cell repertoire and proliferation of germinative cells of the model cestode Mesocestoides corti.

The phylum Platyhelminthes shares a unique population of undifferentiated cells responsible for the proliferation capacity needed for cell renewal, growth, tissue repair and regeneration. These cells have been extensively studied in free-living flatworms, whereas in cestodes the presence of a set of undifferentiated cells, known as germinative cells, has been demonstrated in classical morphology studies, but poorly characterized with molecular biology approaches. Furthermore, several genes have been identified as neoblast markers in free-living flatworms that deserve study in cestode models. Here, different cell types of the model cestode Mesocestoides corti were characterized, identifying differentiated and germinative cells. Muscle cells, tegumental cells, calcareous corpuscle precursor cells and excretory system cells were identified, all of which are non-proliferative, differentiated cell types. Besides those, germinative cells were identified as a population of small cells with proliferative capacity in vivo. Primary cell culture experiments in Dulbecco's Modified Eagle Medium (DMEM), Echinococcus hydatid fluid and hepatocyte conditioned media in non-reductive or reductive conditions confirmed that the germinative cells were the only ones with proliferative capacity. Since several genes have been identified as markers of undifferentiated neoblast cells in free-living flatworms, the expression of pumilio and pL10 genes was analysed by qPCR and in situ hybridization, showing that the expression of these genes was stronger in germinative cells but not restricted to this cell type. This study provides the first tools to analyse and further characterise undifferentiated cells in a model cestode.

Lost and Found: Piwi and Argonaute Pathways in Flatworms.

Platyhelminthes comprise one of the major phyla of invertebrate animals, inhabiting a wide range of ecosystems, and one of the most successful in adapting to parasitic life. Small non-coding RNAs have been implicated in regulating complex developmental transitions in model parasitic species. Notably, parasitic flatworms have lost Piwi RNA pathways but gained a novel Argonaute gene. Herein, we analyzed, contrasted and compared the conservation of small RNA pathways among several free-living species (a paraphyletic group traditionally known as 'turbellarians') and parasitic species (organized in the monophyletic clade Neodermata) to disentangle possible adaptations during the transition to parasitism. Our findings showed that complete miRNA and RNAi pathways are present in all analyzed free-living flatworms. Remarkably, whilst all 'turbellarians' have Piwi proteins, these were lost in parasitic Neodermantans. Moreover, two clusters of Piwi class Argonaute genes are present in all 'turbellarians'. Interestingly, we identified a divergent Piwi class Argonaute in free living flatworms exclusively, which we named 'Fliwi'. In addition, other key proteins of the Piwi pathways were conserved in 'turbellarians', while none of them were detected in Neodermatans. Besides Piwi and the canonical Argonaute proteins, a flatworm-specific class of Argonautes (FL-Ago) was identified in the analyzed species confirming its ancestrallity to all Platyhelminthes. Remarkably, this clade was expanded in parasitic Neodermatans, but not in free-living species. These phyla-specific Argonautes showed lower sequence conservation compared to other Argonaute proteins, suggesting that they might have been subjected to high evolutionary rates. However, key residues involved in the interaction with the small RNA and mRNA cleavage in the canonical Argonautes were more conserved in the FL-Agos than in the Piwi Argonautes. Whether this is related to specialized functions and adaptations to parasitism in Neodermatans remains unclear. In conclusion, differences detected in gene conservation, sequence and structure of the Argonaute family suggest tentative biological and evolutionary diversifications that are unique to Platyhelminthes. The remarkable divergencies in the small RNA pathways between free-living and parasitic flatworms indicate that they may have been involved in the adaptation to parasitism of Neodermatans.

Gene Silencing in the Liver Fluke Fasciola hepatica: RNA Interference.

The chronic infection with the liver fluke of the genus Fasciola spp. is the most prevalent foodborne trematodiasis, affecting at least one-fourth of the world livestock grazing in areas where the parasite is present. Moreover, fascioliasis is considered a major zoonosis mainly in rural areas of central South America, Northern Africa, and Central Asia. Increasing evidences of resistance against triclabendazole may compromise its use as drug of choice; thus, novel control strategies are desperately needed. Functional genomic approaches play a key role in the validation and characterization of new targets for drug and vaccine development. So far, RNA interference has been the only gene silencing approach successfully employed in liver flukes of the genus Fasciola spp. Herein, we describe a detailed step-by-step protocol to perform gene silencing mediated by RNAi in Fasciola hepatica.

Design of a Peptide-Carrier Vaccine Based on the Highly Immunogenic Fasciola hepatica Leucine Aminopeptidase.

Many studies have shown that the degree of organization and repetitiveness of an antigen correlates with its efficiency to induce a B-cell response and production of neutralizing antibodies. Here we describe the design of a chimeric protein based on the hexamer form of the highly immunogenic Fasciola hepatica leucine aminopeptidase as a carrier system of small peptides with potential use as a multiepitope vaccine.

Adaptive Radiation of the Flukes of the Family Fasciolidae Inferred from Genome-Wide Comparisons of Key Species.

Liver and intestinal flukes of the family Fasciolidae cause zoonotic food-borne infections that impact both agriculture and human health throughout the world. Their evolutionary history and the genetic basis underlying their phenotypic and ecological diversity are not well understood. To close that knowledge gap, we compared the whole genomes of Fasciola hepatica, Fasciola gigantica, and Fasciolopsis buski and determined that the split between Fasciolopsis and Fasciola took place ∼90 Ma in the late Cretaceous period, and that between 65 and 50 Ma an intermediate host switch and a shift from intestinal to hepatic habitats occurred in the Fasciola lineage. The rapid climatic and ecological changes occurring during this period may have contributed to the adaptive radiation of these flukes. Expansion of cathepsins, fatty-acid-binding proteins, protein disulfide-isomerases, and molecular chaperones in the genus Fasciola highlights the significance of excretory-secretory proteins in these liver-dwelling flukes. Fasciola hepatica and Fasciola gigantica diverged ∼5 Ma near the Miocene-Pliocene boundary that coincides with reduced faunal exchange between Africa and Eurasia. Severe decrease in the effective population size ∼10 ka in Fasciola is consistent with a founder effect associated with its recent global spread through ruminant domestication. G-protein-coupled receptors may have key roles in adaptation of physiology and behavior to new ecological niches. This study has provided novel insights about the genome evolution of these important pathogens, has generated genomic resources to enable development of improved interventions and diagnosis, and has laid a solid foundation for genomic epidemiology to trace drug resistance and to aid surveillance.

Compositional Analysis of Flatworm Genomes Shows Strong Codon Usage Biases Across All Classes.

In the present work, we performed a comparative genome-wide analysis of 22 species representative of the main clades and lifestyles of the phylum Platyhelminthes. We selected a set of 700 orthologous genes conserved in all species, measuring changes in GC content, codon, and amino acid usage in orthologous positions. Values of 3rd codon position GC spanned over a wide range, allowing to discriminate two distinctive clusters within freshwater turbellarians, Cestodes and Trematodes respectively. Furthermore, a hierarchical clustering of codon usage data differs remarkably from the phylogenetic tree. Additionally, we detected a synonymous codon usage bias that was more dramatic in extreme GC-poor or GC-rich genomes, i.e., GC-poor Schistosomes preferred to use AT-rich terminated synonymous codons, while GC-rich M. lignano showed the opposite behavior. Interestingly, these biases impacted the amino acidic usage, with preferred amino acids encoded by codons following the GC content trend. These are associated with non-synonymous substitutions at orthologous positions. The detailed analysis of the synonymous and non-synonymous changes provides evidence for a two-hit mechanism where both mutation and selection forces drive the diverse coding strategies of flatworms.

Different SNPs in Fasciola hepatica P-glycoprotein from diverse Latin American populations are not associated with Triclabendazole resistance.

The use of Triclabendazole for controlling fasciolosis is compromised by increased drug resistance affecting livestock and humans. Although the mode of action of TCBZ is still unknown, putative candidates and markers of resistance have been advanced. A single nucleotide polymorphism (T687 G) in F. hepatica PGP was proposed as marker of resistance in a small scale study of European susceptible and resistant flukes, but the association was not found in Australian samples. The T687 G SNP was absent in more than 40 samples from 2 TCBZ-resistant and 3 susceptible isolates across Latin America here analyzed. While the American samples showed more variable SNPs than the previous ones, none of the SNPs detected showed a marked association with resistance. Analyzing the 42 kb of the FhPGP gene based on RNAseq data highlights that the variation has been underestimated, suggesting that more detailed efforts are needed in order to identify markers of resistance.

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

Pleiotropic alterations in gene expression in Latin American Fasciola hepatica isolates with different susceptibility to drugs.

BACKGROUND: Fasciola hepatica is the main agent of fasciolosis, a zoonotic disease affecting livestock worldwide, and an emerging food-borne disease in humans. Even when effective treatments are available, drugs are costly and can result in tolerance, liver damage and normally they do not prevent reinfection. Drug-resistant strains in livestock have been reported in various countries and, more worryingly, drug resistance in human cases has emerged in South America. The present study aims to characterize the transcriptome of two South American resistant isolates, the Cajamarca isolate from Peru, resistant to both triclabendazole and albendazole (TCBZR/ABZR) and the Rubino isolate from Uruguay, resistant to ABZ (TCBZS/ABZR), and compare them to a sensitive strain (Cenapa, Mexico, TCBZS/ABZS) to reveal putative molecular mechanisms leading to drug resistance. RESULTS: We observed a major reduction in transcription in the Cajamarca TCBZR/ABZR isolate in comparison to the other isolates. While most of the differentially expressed genes are still unannotated, several trends could be detected. Specific reduction in the expression levels of cytoskeleton proteins was consistent with a role of tubulins as putative targets of triclabendazole (TCBZ). A marked reduction of adenylate cyclase might be underlying pleiotropic effects on diverse metabolic pathways of the parasite. Upregulation of GST mu isoforms suggests this detoxifying mechanism as one of the strategies associated with resistance. CONCLUSIONS: Our results stress the value of transcriptomic approaches as a means of providing novel insights to advance the understanding of drug mode of action and drug resistance. The results provide evidence for pleiotropic variations in drug-resistant isolates consistent with early observations of TCBZ and ABZ effects and recent proteomic findings.

Expression, purification and characterization of two leucine aminopeptidases of the blood fluke, Schistosoma mansoni.

Schistosomiasis is a major neglected tropical disease (NTD) and considered the most important of the human helminthiases in terms of morbidity and mortality. Whereas treatment with praziquantel has been effective since the 1980s, the potential for the emergence of drug resistance has propelled the search for new interventions. Studies have revealed key roles of proteases in parasitic helminths during establishment of infection, tissue invasion, immune evasion, parasite feeding and development throughout the different developmental stages, pinpointing them as possible candidates. The leucine aminopeptidases (LAPs), members of the M17 family of Zn-metalloproteases, preferentially cleave leucine (Leu) residues at the N-terminal end of proteins and short peptides. These enzymes display broad proteolytic activities beyond Leu hydrolysis and are involved in processing, maturation, activation and/or degradation of substrates. As a vaccine immunogen, LAP induces protection against infection with the liver fluke Fasciola hepatica. Herein, two LAPs, SmLAP1 (Smp_030000) and SmLAP2 (Smp_083870) of the human blood fluke Schistosoma mansoni were cloned, expressed, purified and biochemically characterized. The enzymes differed in activity against diagnostic substrates, including leucine, methionine and arginine, with an optimal pH of 8.0. The activity increased in the presence of Mg+2 and Mn+2, and was inhibited by bestatin, a specific inhibitor of aminopeptidase. In addition, 1,10-phenanthroline and EDTA inhibited the enzymatic activity of SmLAP2. Finally, immunolocalization using antibodies specific for SmLAP1 and SmLAP2 identified the expression of these proteases in the egg and adult developmental stages of S. mansoni, and in intestinal epithelia, vitelline cells and sub-tegumental regions of the parasite. Characterization of schistosome proteases not only enhances understanding of the biology of schistosomes and schistosomiasis, but may also provide novel intervention approaches.

Conservation and diversification of small RNA pathways within flatworms.

BACKGROUND: Small non-coding RNAs, including miRNAs, and gene silencing mediated by RNA interference have been described in free-living and parasitic lineages of flatworms, but only few key factors of the small RNA pathways have been exhaustively investigated in a limited number of species. The availability of flatworm draft genomes and predicted proteomes allowed us to perform an extended survey of the genes involved in small non-coding RNA pathways in this phylum. RESULTS: Overall, findings show that the small non-coding RNA pathways are conserved in all the analyzed flatworm linages; however notable peculiarities were identified. While Piwi genes are amplified in free-living worms they are completely absent in all parasitic species. Remarkably all flatworms share a specific Argonaute family (FL-Ago) that has been independently amplified in different lineages. Other key factors such as Dicer are also duplicated, with Dicer-2 showing structural differences between trematodes, cestodes and free-living flatworms. Similarly, a very divergent GW182 Argonaute interacting protein was identified in all flatworm linages. Contrasting to this, genes involved in the amplification of the RNAi interfering signal were detected only in the ancestral free living species Macrostomum lignano. We here described all the putative small RNA pathways present in both free living and parasitic flatworm lineages. CONCLUSION: These findings highlight innovations specifically evolved in platyhelminths presumably associated with novel mechanisms of gene expression regulation mediated by small RNA pathways that differ to what has been classically described in model organisms. Understanding these phylum-specific innovations and the differences between free living and parasitic species might provide clues to adaptations to parasitism, and would be relevant for gene-silencing technology development for parasitic flatworms that infect hundreds of million people worldwide.

Genomes of Fasciola hepatica from the Americas Reveal Colonization with Neorickettsia Endobacteria Related to the Agents of Potomac Horse and Human Sennetsu Fevers.

Food borne trematodes (FBTs) are an assemblage of platyhelminth parasites transmitted through the food chain, four of which are recognized as neglected tropical diseases (NTDs). Fascioliasis stands out among the other NTDs due to its broad and significant impact on both human and animal health, as Fasciola sp., are also considered major pathogens of domesticated ruminants. Here we present a reference genome sequence of the common liver fluke, Fasciola hepatica isolated from sheep, complementing previously reported isolate from cattle. A total of 14,642 genes were predicted from the 1.14 GB genome of the liver fluke. Comparative genomics indicated that F. hepatica Oregon and related food-borne trematodes are metabolically less constrained than schistosomes and cestodes, taking advantage of the richer millieux offered by the hepatobiliary organs. Protease families differentially expanded between diverse trematodes may facilitate migration and survival within the heterogeneous environments and niches within the mammalian host. Surprisingly, the sequencing of Oregon and Uruguay F. hepatica isolates led to the first discovery of an endobacteria in this species. Two contigs from the F. hepatica Oregon assembly were joined to complete the 859,205 bp genome of a novel Neorickettsia endobacterium (nFh) closely related to the etiological agents of human Sennetsu and Potomac horse fevers. Immunohistochemical studies targeting a Neorickettsia surface protein found nFh in specific organs and tissues of the adult trematode including the female reproductive tract, eggs, the Mehlis' gland, seminal vesicle, and oral suckers, suggesting putative routes for fluke-to-fluke and fluke-to-host transmission. The genomes of F. hepatica and nFh will serve as a resource for further exploration of the biology of F. hepatica, and specifically its newly discovered trans-kingdom interaction with nFh and the impact of both species on disease in ruminants and humans.

Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach.

BACKGROUND: Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections. METHODOLOGY/PRINCIPLE FINDINGS: We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1). Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 µM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells. CONCLUSIONS/SIGNIFICANCE: Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34) is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues for the development of novel agents to control fluke infection and possibly other helminthic diseases.

The miRnome of Fasciola hepatica juveniles endorses the existence of a reduced set of highly divergent micro RNAs in parasitic flatworms.

The liver fluke Fasciola hepatica is a foodborne zoonotic parasite affecting livestock worldwide, with increasing relevance in human health. The first developmental stage that the host meets after ingestion of the parasite is the newly excysted juvenile, that actively transverses the gut wall and migrates to its final location in the liver. The regulation of the early developmental events in newly excysted juveniles is still poorly understood and a relevant target for control strategies. Here we investigated the putative involvement of small regulatory RNAs in the invasion process. The small RNA population of the newly excysted juvenile fall into two classes, one represented by micro (mi)RNAs and a secondary group of larger (32-33 nucleotides) tRNA-derived sequences. We identified 40 different miRNAs, most of those belonging to ancient miRNAs conserved in protostomes and metazoans, notably with a highly predominant miR-125b variant. Remarkably, several protostomian and metazoan conserved families were not detected in consonance with previous reports of drastic miRnome reduction in parasitic flatworms. Additionally, a set of five novel miRNAs was identified, probably associated with specific gene regulation expression needs in F. hepatica. While sequence conservation in mature miRNA is high across the metazoan tree, we observed that flatworm miRNAs are more divergent, suggesting that mutation rates in parasitic flatworms could be high. Finally, the distinctive presence of tRNA-derived sequences, mostly 5' tRNA halves of selected tRNAs in the small RNA population of newly excysted juveniles, raises the possibility that both miRNA and tRNA fragments participate in the regulation of gene expression in this parasite.

RNA interference in Fasciola hepatica newly excysted juveniles: long dsRNA induces more persistent silencing than siRNA.

In trematodes RNA interference is the current tool of choice for functional analysis of genes since classical reverse genetic approaches remain unavailable. Whereas this approach has been optimized in schistosomes, few reports are available for other trematodes, likely reflecting the difficulties in the establishment of the technology. Here we standardized conditions for RNAi in the liver fluke Fasciola hepatica, the causative agent of fasciolosis, one of the most problematic infections affecting livestock worldwide. Targeting a single copy gene, encoding leucine aminopeptidase (LAP) as a model, we refined delivery conditions which identified electro-soaking, i.e. electroporation and subsequent incubation as efficient for introduction of small RNAs into the fluke. Knock down of LAP was achieved with as little as 2.5 µg/ml dsRNA concentrations, which may reduce or obviate off-target effects. However, at these concentrations, tracking incorporation by fluorescent labeling was difficult. While both long dsRNA and short interfering RNA (siRNA) are equally effective at inducing a short-term knock down, dsRNA induced persistent silencing up to 21 days after treatment, suggesting that mechanisms of amplification of the interfering signal can be present in this pathogen. Persistent silencing of the invasive stage for up to 3 weeks (close to what it takes for the fluke to reach the liver) opens the possibility of using RNAi for the validation of putative therapeutic targets.

Dissecting the active site of the collagenolytic cathepsin L3 protease of the invasive stage of Fasciola hepatica.

BACKGROUND: A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2. METHODOLOGY/PRINCIPAL FINDINGS: Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3. CONCLUSIONS/SIGNIFICANCE: These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases.

Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.

Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. DATABASE ACCESSION: Sequence data from this study have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/embl) under accession number ERP000379.

An antibiotic selection marker for schistosome transgenesis.

Drug selection is widely used in transgene studies of microbial pathogens, mammalian cell and plant cell lines. Drug selection of transgenic schistosomes would be desirable to provide a means to enrich for populations of transgenic worms. We adapted murine leukaemia retrovirus vectors - widely used in human gene therapy research - to transduce schistosomes, leading to integration of transgenes into the genome of the blood fluke. A dose-response kill curve and lethal G418 (geneticin) concentrations were established: 125-1,000µg/ml G418 were progressively more toxic for schistosomules of Schistosoma mansoni with toxicity increasing with antibiotic concentration and with duration of exposure. By day 6 of exposure to ⩾500µg/ml, significantly fewer worms survived compared with non-exposed controls and by day 8, significantly fewer worms survived than controls at ⩾250µg/ml G418. When schistosomules were transduced with murine leukaemia retrovirus encoding the neomycin resistance (neoR) transgene and cultured in media containing G418, the neoR transgene rescued transgenic schistosomules from the antibiotic; by day 4 in 1,000µg/ml and by day 8 in 500µg/ml G418, significantly more transgenic worms survived the toxic effects of the antibiotic. More copies of neoR were detected per nanogram of genomic DNA from populations of transgenic schistosomes cultured in G418 than from transgenic schistosomes cultured without G418. This trend was G418 dose-dependent, demonstrating enrichment of transgenic worms from among the schistosomules exposed to virions. Furthermore, higher expression of neoR was detected in transgenic schistosomes cultured in the presence of G418 than in transgenic worms cultured without antibiotic. The availability of antibiotic selection can be expected to enhance progress with functional genomics research on the helminth parasites responsible for major neglected tropical diseases.