The Sinbad retrotransposon from the genome of the human blood fluke, Schistosoma mansoni, and the distribution of related Pao-like elements

BACKGROUND: Of the major families of long terminal repeat (LTR) retrotransposons, the Pao/BEL family is probably the least well studied. It is becoming apparent that numerous LTR retrotransposons and other mobile genetic elements have colonized the genome of the human blood fluke, Schistosoma mansoni. RESULTS: A proviral form of Sinbad, a new LTR retrotransposon, was identified in the genome of S. mansoni. Phylogenetic analysis indicated that Sinbad belongs to one of five discreet subfamilies of Pao/BEL like elements. BLAST searches of whole genomes and EST databases indicated that members of this clade occurred in species of the Insecta, Nematoda, Echinodermata and Chordata, as well as Platyhelminthes, but were absent from all plants, fungi and lower eukaryotes examined. Among the deuterostomes examined, only aquatic species harbored these types of elements. All four species of nematode examined were positive for Sinbad sequences, although among insect and vertebrate genomes, some were positive and some negative. The full length, consensus Sinbad retrotransposon was 6,287 bp long and was flanked at its 5'- and 3'-ends by identical LTRs of 386 bp. Sinbad displayed a triple Cys-His RNA binding motif characteristic of Gag of Pao/BEL-like elements, followed by the enzymatic domains of protease, reverse transcriptase (RT), RNAseH, and integrase, in that order. A phylogenetic tree of deduced RT sequences from 26 elements revealed that Sinbad was most closely related to an unnamed element from the zebrafish Danio rerio and to Saci-1, also from S. mansoni. It was also closely related to Pao from Bombyx mori and to Ninja of Drosophila simulans. Sinbad was only distantly related to the other schistosome LTR retrotransposons Boudicca, Gulliver, Saci-2, Saci-3, and Fugitive, which are gypsy-like. Southern hybridization and bioinformatics analyses indicated that there were about 50 copies of Sinbad in the S. mansoni genome. The presence of ESTs representing transcripts of Sinbad in numerous developmental stages of S. mansoni along with the identical 5'- and 3'-LTR sequences suggests that Sinbad is an active retrotransposon. CONCLUSION: Sinbad is a Pao/BEL type retrotransposon from the genome of S. mansoni. The Pao/BEL group appears to be comprised of at least five discrete subfamilies, which tend to cluster with host species phylogeny. Pao/BEL type elements appear to have colonized only the genomes of the Animalia. The distribution of these elements in the Ecdysozoa, Deuterostomia, and Lophotrochozoa is discontinuous, suggesting horizontal transmission and/or efficient elimination of Pao-like mobile genetic elements from some genomes

Granulin Secreted by the Food-Borne Liver Fluke Opisthorchis viverrini Promotes Angiogenesis in Human Endothelial Cells

The liver fluke Opisthorchis viverrini is a food-borne, zoonotic pathogen endemic to Thailand and adjacent countries in Southeast Asia. The adult developmental stage of the O. viverrini parasite excretes and secretes numerous proteins within the biliary tract including the gall bladder. Lesions caused by the feeding activities of the liver fluke represent wounds that undergo protracted cycles of healing and re-injury during chronic infection, which can last for decades. Components of the excretory/secretory (ES) complement released by the worms capably drive proliferation of bile duct epithelial cells and are implicated in establishing the oncogenic milieu that leads to bile duct cancer, cholangiocarcinoma. An ES protein, the secreted granulin-like growth factor termed Ov-GRN-1, accelerates wound resolution in mice and in vitro. To investigate angiogenesis (blood vessel development) that may contribute to wound healing promoted by liver fluke granulin and, by implication, to carcinogenesis during chronic opisthorchiasis, we employed an in vitro tubule formation assay (TFA) where human umbilical vein endothelial cells were grown on gelled basement matrix. Ten and 40 nM Ov-GRN-1 significantly stimulated angiogenesis as monitored by cellular proliferation and by TFA in real time. This demonstration of potent angiogenic property of Ov-GRN-1 bolsters earlier reports on the therapeutic potential for chronic non-healing wounds of diabetics, tobacco users, and the elderly and, in addition, showcases another of the hallmark of cancer characteristic of this carcinogenic liver fluke

Granulin Secreted by the Food-Borne Liver Fluke

The liver fluke Opisthorchis viverrini is a food-borne, zoonotic pathogen endemic to Thailand and adjacent countries in Southeast Asia. The adult developmental stage of the O. viverrini parasite excretes and secretes numerous proteins within the biliary tract including the gall bladder. Lesions caused by the feeding activities of the liver fluke represent wounds that undergo protracted cycles of healing and re-injury during chronic infection, which can last for decades. Components of the excretory/secretory (ES) complement released by the worms capably drive proliferation of bile duct epithelial cells and are implicated in establishing the oncogenic milieu that leads to bile duct cancer, cholangiocarcinoma. An ES protein, the secreted granulin-like growth factor termed Ov-GRN-1, accelerates wound resolution in mice and in vitro. To investigate angiogenesis (blood vessel development) that may contribute to wound healing promoted by liver fluke granulin and, by implication, to carcinogenesis during chronic opisthorchiasis, we employed an in vitro tubule formation assay (TFA) where human umbilical vein endothelial cells were grown on gelled basement matrix. Ten and 40 nM Ov-GRN-1 significantly stimulated angiogenesis as monitored by cellular proliferation and by TFA in real time. This demonstration of potent angiogenic property of Ov-GRN-1 bolsters earlier reports on the therapeutic potential for chronic non-healing wounds of diabetics, tobacco users, and the elderly and, in addition, showcases another of the hallmark of cancer characteristic of this carcinogenic liver fluke

Electroporation Facilitates Introduction of Reporter Transgenes and Virions into Schistosome Eggs

The genome sequences of two of the three major species of schistosomes are now available. Molecular tools are needed to determine the importance of these new genes. With this in mind, we investigated introduction of reporter transgenes into schistosome eggs, with the longer-term aim of manipulation of schistosome genes and gene functions. The egg is a desirable developmental stage for genome manipulation, not least because it contains apparently accessible germ cells. Introduction of transgenes into the germ cells of schistosome eggs might result in transgenic schistosomes. However, the egg is surrounded by a thick shell which might block access to entry of transgenes. We cultured eggs in the presence of three types of reporter transgenes of increasing molecular size, and in addition we tried to produce transient holes in the eggs by electroporation to investigate whether the transgenes would more easily enter the eggs. Electroporation of eggs appeared to allow entry of two larger types of transgenes into cultured schistosome eggs, messenger RNA encoding firefly luciferase, and retroviral virions. We anticipate that this approach, electroporation of transgenes into schistosome eggs, will facilitate genetic manipulation of schistosomes for investigating the importance of schistosome genes and gene products as new intervention targets

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\u27 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

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, \u3e10-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

Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds

Liver fluke granulin promote extracellular vesicle-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition

HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni.

Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; \u3e8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate