This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the “96 Years of Opisthorchiasis. International Congress of Liver Flukes”. It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor.
Although draft genome sequences of two of the major human schistosomes, Schistosoma japonicum and S. mansoni are available, the structures and characteristics of most genes and the influence of exogenous genes on the metabolism of schistosomes remain uncharacterized. Furthermore, which functional genomics approaches will be tractable for schistosomes are not yet apparent. Here, the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped pantropic retroviral vector pBABE-puro was modified to incorporate the human telomerase reverse transcriptase gene (hTERT) as a reporter, under the control of the retroviral long terminal repeat (LTR). Pseudotyped virions were employed to transduce S. japonicum to investigate the utility of retrovirus-mediated transgenesis of S. japonicum and the activity of human telomerase reverse transcriptase as a reporter transgene in schistosomes. Schistosomules perfused from experimentally infected rabbits were cultured for six days after exposure to the virions after which genomic DNAs from virus-exposed and control worms were extracted. Analysis of RNA from transduced parasites and immunohistochemistry of thin parasite sections revealed expression of hTERT in the transduced worms. Expression of hTERT was also confirmed by immunoblot analysis. These findings indicated that S. japonicum could be effectively transduced by VSVG pseudotyped retrovirus carrying the hTERT gene. Given the potential of hTERT to aid in derivation of immortalized cells, these findings suggest that this pantropic retroviral approach can be employed to transduce cells from specific tissues and organs of schistosomes to investigate the influence of transgene hTERT on growth and proliferation of schistosome cells.
Schistosoma japonicum; glycoprotein of vesicular stomatitis virus (VSVG); pantropic retrovirus; murine leukemia virus; transgene; telomerase
Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and adjacent countries. In addition to infection-associated morbidity, infection with O. viverrini and the related Clonorchis sinensis are unarguable risk factors for cholangiocarcinoma, bile duct cancer. Here we review the pathogenesis of opisthorchiasis and the association of O. viverrini infection and bile duct cancer, focusing on the molecular parallels between wound healing, chronic inflammation and cancer development. We review a schema for human disease progression from fluke infection, chronic opisthorchiasis, advanced periductal fibrosis, and cholangiocarcinogenesis, and present a rationale for biomarker discovery to facilitate early intervention. We conclude by addressing post-genomic advances with a view to developing new control strategies to combat this infectious cancer.
Opisthorchis; liver fluke; cholangiocarcinoma; liver cancer; granulin; periductal fibrosis; excretory/secretory; wound healing
Schistosoma japonicum is a parasitic flatworm that causes human schistosomiasis, a significant cause of morbidity in China and the Philippines. Here we present a draft genomic sequence for the worm, which is the first reported for any flatworm, indeed for the superphylum Lophotrochozoa. The genome provides a global insight into the molecular architecture and host interaction of this complex metazoan pathogen, revealing that it can exploit host nutrients, neuroendocrine hormones and signaling pathways for growth, development and maturation. Having a complex nervous system and a well developed sensory system, S. japonicum can accept stimulation of the corresponding ligands as a physiological response to different environments, such as fresh water or the tissues of its intermediate and mammalian hosts. Numerous proteinases, including cercarial elastase, are implicated in mammalian skin penetration and haemoglobin degradation. The genomic information will serve as a valuable platform to facilitate development of new interventions for schistosomiasis control.
Throughout Southeast Asia there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium), particularly in people from rural settings in Laos and Northeast Thailand who are infected with the liver fluke, Opisthorchis viverrini, one of only three carcinogenic eukaryotic pathogens. More ubiquitous carcinogenic microbes, such as Helicobacter pylori, induce cancer in less than 1% of infected people, while as many as one-sixth of people with opisthorchiasis will develop CCA. The mechanisms by which O. viverrini causes cancer are multi-factorial, involving mechanical irritation from the activities and movements of the flukes, immunopathology, dietary nitrosamines and the secretion of parasite proteins that promote a tumourigenic environment. Genomic and proteomic studies of the liver fluke secretome have accelerated the discovery of parasite proteins with known/potential roles in pathogenesis and tumourigenesis, establishing a framework towards understanding, and ultimately preventing, the morbidity and mortality attributed to this highly carcinogenic parasite.
Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.
transgenesis; transposon; piggyBac; retrovirus; integration; vector based RNAi
A novel 22.8 kDa of Opisthorchis viverrini (Ov) calcium-binding EF-hand protein (Ov CaBP) was identified and isolated from an immunoscreening of the adult stage Ov cDNA library by using a human cholangiocarcinoma (CCA) serum. This protein was related to other calcium binding proteins and conserved among the trematodes. Ov CaBP shared 98% amino acid identity to 22.8 kDa of Clonorchis sinensis CaBP and both were classified as a new group of CaBP EF-hand protein by multiple sequence alignment and phylogenetic tree analysis. The open reading frame of Ov CaBP was 585 bp which encoded for 194 amino acids. The N-terminal part is composed of two calcium-binding EF-hand motifs whereas the C-terminal part contains a dynein light chain motif (DLC). In addition, transcription analysis by RT-PCR revealed that it was constitutively transcribed in all stages, including metacercariae, juvenile, and adult. Furthermore, recombinant Ov CaBP protein (rOv CaBP) was expressed as a soluble protein and antibody generated against this rOv CaBP protein was capable of detecting Ov CaBP in the Ov somatic extracts but not in Ov ES products. This anti-rOv CaBP serum was also used to localize Ov CaBP in Ov infected hamster’s liver sections which the distribution of Ov CaBP was located in gut epithelium, miracidia in eggs and slightly in parenchyma. Moreover, rOv CaBP protein showed a calcium binding property in non-denaturing gel mobility shift assay.
Opisthorchis viverrini; calcium-binding EF-hand; protein expression; gel mobility shift assay; immunolocalization
The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilising systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1at mRNA and protein levels were observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemsitry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation.
liver fluke; Opisthorchis viverrini; thioredoxin; redox system; antioxidant
Draft genome sequences for Schistosoma japonicum and S. mansoni are now available. The schistosome genome encodes ~13000 protein-encoding genes for which the functions of few are well understood. Nonetheless, the new genes represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past 15 years, noteworthy progress has been achieved towards development of tools for gene manipulation and transgenesis of schistosomes. A brief history of genetic manipulation is presented, along with a review of the field with emphasis on reports of integration of transgenes into schistosome chromosomes.
Schistosomes; genetic manipulation; transgenesis; chromosome integration; germ line; retrovirus; murine leukaemia virus; pseudotyped gammaretrovirus; transposon; piggyBac
The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for many years. The mechanisms by which it avoids oxidative damage are unknown. In this study, thioredoxin peroxidase (TPx), a member of the peroxiredoxin superfamily, was cloned from an O. viverrini cDNA library. O. viverrini TPx cDNA encoded a polypeptide of 212 amino acid residues, of molecular mass 23.57 kDa. The putative amino acid sequence shared 60-70% identity with TPXs from other helminths and from mammals, and phylogenetic analysis revealed a close relationship between TPxs from O. viverrini and other trematodes. Recombinant O. viverrini TPx was expressed as soluble protein in Escherichia coli. The recombinant protein dimerized, and its antioxidant activity was deduced by observing protection of nicking of supercoiled plasmid DNA by hydroxyl radicals. Antiserum raised against O. viverrini TPx recognized native proteins from egg, metacercaria and adult developmental stages of the liver fluke and excretory-secretory products released by adult O. viverrini. Immunolocalization studies revealed ubiquitous expression of TPx in O. viverrini organs and tissues. TPx was also detected in bile fluid and bile duct epithelial cells surrounding the flukes two weeks after infection of hamsters with O. viverrini. In addition, TPx was observed in the secondary (small) bile ducts where flukes cannot reach due to their large size. These results suggested that O. viverrini TPx plays a significant role in protecting the parasite against damage induced by reactive oxygen species from inflammation.
liver fluke; Opisthorchis viverrini; peroxiredoxin; thioredoxin peroxidase; thiol-specific antioxidant; antioxidant enzyme
Functional genomics have not been reported for Opisthorchis viverrini or the related fish-borne fluke, Clonorchis sinensis. Here we describe the introduction by square wave electroporation of Cy3-labelled small RNA into adult O. viverrini worms. Adult flukes were subjected to square wave electroporation employing a single pulse for 20 ms of 125 volts in the presence of 50 μg/ml of Cy3-siRNA. The parasites tolerated this manipulation and, at 24 and 48 hours after electroporation, fluorescence from the Cy3-siRNA was evident throughout the parenchyma of the worms, with strong fluorescence evident in the guts and reproductive organs of the adult worms. Second, other worms were treated using the same electroporation settings with double stranded RNA targeting an endogenous papain-like cysteine protease, cathepsin B. This manipulation resulted in a significant reduction in specific mRNA levels encoding cathepsin B, and a significant reduction in cathepsin B activity against the diagnostic peptide, Z-Arg-Arg-AMC. This appears to be the first report of introduction of reporter genes into O. viverrini and the first report of experimental RNA interference (RNAi) in this fluke. The findings indicated the presence of an intact RNAi pathway in these parasites which, in turn, provides an opportunity to probe gene functions in this neglected tropical disease pathogen.
O. viverrini; RNA interference; cathepsin B; liver fluke
More than 750 million people are at risk of infection with food-borne liver flukes. Opisthorchis viverrini is considered among the most important of these parasites, due to its strong association with cholangiocarcinoma (CCA). O. viverrini infection results in a chronic inflammatory challenge to the host, which can lead to advanced, pathogen-specific disease sequelae including obstructive jaundice, hepatomegaly, cholecystitis, as well as CCA. However, before disease sequelae are apparent, important inflammatory changes to the liver can be detected early during O. viverrini infection. In a case-control study involving 328 men and women with O. viverrini infection, we determined the presence of advanced periductal fibrosis in asymptomatic, O. viverrini-infected individuals and then measured cytokine responses to O. viverrini excretory/secretory products (ES). In the 200 participants with advanced periductal fibrosis (cases), levels of Interleukin (IL)-6 to O. viverrini ES were 8 times higher than levels of the 128 O. viverrini-infected individuals without advanced periductal fibrosis (controls). Moreover, elevated IL-6 to parasite ES was associated with increased risk of advanced periductal fibrosis by 63% in a model adjusted for sex and age. The risk of advanced periductal fibrosis was also found to increase with higher levels of IL-6: individuals in the third quartile of IL-6-ES production had a 127% higher risk of developing advanced periductal fibrosis than individuals in the first quartile of IL-6 production. O. viverrini-infected individuals with advanced periductal fibrosis showed other hepatobiliary abnormalities, including reduced gallbladder contractility and the presence of gallbladder sludge.
These data strongly implicate a role for parasite specific IL-6 in the pathogenesis of advanced periductal fibrosis in opisthorchiasis, with possible links to other hepatobiliary abnormalities, including cholangiocarcinoma.
chronic inflammation; opisthorchiasis; cytokine; periductal fibrosis; case-control study
Blood flukes or schistosomes are the causative agents of human schistosomiasis, one of the major neglected tropical diseases. Draft genome sequences have been reported for schistosomes, but functional genomics tools are needed to investigate the role and essentiality of the newly reported genes. Vector based RNA interference can contribute to functional genomics analysis for schistosomes. Using mRNA encoding reporter firefly luciferase as a model target, we compared the performance of a schistosome and a human promoter from the U6 gene in driving shRNA in human fibrosarcoma cells and in cultured schistosomes. Further, both a retroviral (Murine leukemia virus [MLV]) and plasmid (piggyBac, pXL-Bac II) vector were utilized. The schistosome U6 gene promoter was 270 bp in length, the human U6 gene promoter was 264 bp; they shared 41% identity. Following transduction of both HT1080 fibrosarcoma cells and schistosomules of Schistosoma mansoni with pseudotyped MLV virions, stronger knockdown of luciferase activity was seen with the virions encoding the human U6 promoter driven shRNA than the schistosome U6 promoter. A similar trend was seen after transfection of HT1080 cells and schistosomules with the pXL-Bac-II constructs – stronger knockdown of luciferase activity was seen with constructs encoding the human compared to schistosome U6 promoter. The findings indicate that a human U6 gene promoter drives stronger shRNA activity than its schistosome orthologue, not only in a human cancer cell line but also in larval schistosomes. This RNA polymerase III promoter represents a potentially valuable component for vector based RNA interference studies in schistosomes and related platyhelminth parasites.
Schistosome; Schistosoma mansoni; short hairpin RNA; U6 gene; promoter; firefly luciferase; RNA interference; murine leukemia virus; piggyBac transposon
Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken β-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5′- and 3′-Long Terminal Repeats (LTRs) flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3′-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3′-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed two to 20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference.
Schistosome; pseudotyped retrovirus; cHS4 insulator; transgene
Treatment for clinical schistosomiasis has relied centrally on the broad spectrum anthelmintic praziquantel; however, there is limited information on its mode of action or the molecular response of the parasite. This paper presents a transcriptional and functional approach to defining the molecular responses of schistosomes to praziquantel. Differential gene expression in Schistosoma japonicum was investigated by transcriptome-wide microarray analysis of adult worms perfused from infected mice after 0.5 to 24 hours after oral administration of sub-lethal doses of praziquantel. Genes up-regulated initially in male parasites were associated with “Tegument/Muscle Repair” and “Lipid/Ion Regulation” functions and were followed by “Drug Resistance” and “Ion Regulation” associated genes. Prominent responses induced in female worms included up-regulation of “Ca2+ Regulation” and “Drug Resistance” genes and later by transcripts of “Detoxification” and “Pathogen Defense” mechanisms. A subset of highly over-expressed genes, with putative drug resistance/detoxification roles or Ca2+-dependant/modulatory functions, were validated by qPCR. The leading candidate among these was CamKII, a putative calcium/calmodulin-dependent protein kinase type II delta chain. RNA interference was employed to knockdown CamKII in S. japonicum to determine the role of CamKII in the response to praziquantel. After partial-knockdown, schistosomes were analysed using IC50 concentrations (50% worm motility) and quantitative monitoring of parasite movement. When CamKII transcription was reduced by 50–69% in S. japonicum, the subsequent effect of an IC50 dosage of praziquantel was exacerbated, reducing motility from 47% to 27% in female worms and from 61% to 23% in males. These observations indicated that CamKII mitigates the effects of praziquantel, probably through stabilising Ca2+ fluxes within parasite muscles and tegument. Together, these studies comprehensively charted transcriptional changes upon exposure to praziquantel and, notably, identified CamKII as potentially central to the, as yet undefined, mode of action of praziquantel.
Schistosome infected mice were treated orally with a single sub-lethal dose of the anthelmintic praziquantel. Parasites were subsequently isolated 0.5–24 hours later and analysed by gene expression microarray. The transcriptional pattern was influenced by the sex of the parasite. Males up-regulated genes associated with surface integrity, muscle function, ion regulation and drug resistance. While for female worms, genes associated with calcium regulation, drug resistance, detoxification and pathogen defense were transcriptionally elevated. Based on these findings, genes with putative ion homeostasis or drug metabolism roles were further investigated for their prospective roles praziquantel action, using the gene silencing procedure RNA interference. The leading gene was CamKII (calcium/calmodulin-dependent protein kinase type II), a member of the Calcium Signalling Pathway. A quantitative assay for parasite motility during praziquantel exposure was used to study worms in which the CamKII gene had been partially silenced. When CamKII transcription was reduced, the effects of praziquantel were augmented, visualised by reduced parasite movement. These functional studies indicate a key role for the kinase CamKII in the mode of action of the praziquantel, and suggest that future studies on this enzyme and other calcium signaling pathway components will be constructive in understanding how praziquantel kills schistosomes.
Cholangiocarcinoma (CCA) – bile duct cancer - is associated with late presentation, poses challenges for diagnosis, and has high mortality. These features t highlight the desperate need for biomarkers than can be measured early and in accessible body fluids such as plasma of people at risk for developing this lethal cancer. In this manuscript, we address previous limitations in the discovery stage of biomarker(s) for CCA and indicate how new generation of “omics’ technologies could be used for biomarker discovery in Thailand. A key factor in the success of this biomarker program for CCA is the combination of cutting edge technology with strategic sample acquisition by a biorepositories.
Biomarkers; cholangiocarcinoma; liver fibrosis; Opisthorchiasis
A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis.
Opisthorchiasis; cathepsin B; ELISA; immunodiagnosis; serodiagnosis; cysteine protease; Thailand; cholangiocarcinoma
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 leukemia retrovirus (MLV) 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 to 1,000 μg/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 ≥500 μg/ml, significantly fewer worms survived compared with non-exposed controls and by day 8, significantly fewer worms survived than controls at ≥250 μg/ml G418. When schistosomules were transduced with MLV 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 1,000 μg/ml and by day 8 in 500 μg/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.
Schistosome; Transgene; Antibiotic selection; Neomycin; G418; Geneticin; Retrovirus; neoR
The human liver fluke, Opisthorchis viverrini, is designated as a group 1 carcinogen, and is the major risk factor for cholangiocarcinoma in endemic countries throughout Southeast Asia. Proteins in the excretory-secretory products and tegumental surface membranes of the fluke have been proposed to play pivotal roles in parasite survival in the host, and subsequent pathogenesis. These macromolecules are therefore valid targets for the development of vaccines and new drugs to control the infection. Tetraspanins (TSP) are prominent components of the tegument of blood flukes where they are essential for tegument formation, are directly exposed to the immune system, and are major targets for a schistosomiasis vaccine. We propose that similar molecules in the surface membranes of O. viverrini are integral to tegument biogenesis and will be efficacious vaccine antigens.
The cDNA sequence encoding O. viverrini tetraspanin-1 (Ov-TSP-1) was identified and cloned. The Ov-tsp-1gene was isolated from a cDNA library. Ov-tsp-1 mRNA was expressed most highly in metacercariae and eggs, and to a lesser extent in juvenile and adult worms. Immunolocalization with adult flukes confirmed that Ov-TSP-1 was expressed in the tegument and eggs in utero. Western blot analysis of rOv-TSP-1 probed with sera from O. viverrini-infected humans and hamsters indicated that both hosts raise antibody responses against the native TSP. Using RNA interference we silenced the expression level of Ov-tsp-1 mRNA in adult flukes by up to 72% by 10 days after delivery of dsRNA. Ultrastructural morphology of adult worms treated with Ov-tsp-1 dsRNA displayed a distinctly vacuolated and thinner tegument compared with controls.
This is the first report of a tetraspanin from the tegument of a liver fluke. Our data imply that tetraspanins play important structural roles in the development of the tegument in the adult fluke. Potential uses of O. viverrini tetraspanins as novel interventions are discussed.
Liver fluke infection is a fish borne disease that afflicts millions of residents in Thailand and Laos. Infection results from eating undercooked freshwater fish contaminated with larvae of the worm Opisthorchis viverrini. Infection can lead to cancer of the bile ducts (cholangiocarcinoma). Indeed, O. viverrini is designated as a Group 1 carcinogen by the World Health Organization, i.e. a definitive cause for cancer. Proteins produced at the surface and/or released from this parasite play pivotal roles in maintaining the infection and disease. These proteins are valid targets for development of vaccines and new drugs. Tetraspanins are prominent in the tegument (the surface covering) of parasites closely related to O. viverrini where they are exposed to immune responses. Similar molecules on the surface of O. viverrini may be vital for the parasite's survival and may make effective vaccines. Here the gene coding for O. viverrini tetraspanin-1 (Ov-TSP-1) was investigated. We used electron microscopy to show that Ov-TSP-1 is expressed in the tegument. We then silenced expression of the gene encoding Ov-TSP-1 and showed that this resulted in malformation of the tegument, highlighting the importance of this molecule for parasite development and its potential as a vaccine target.
The majority of the world's population now lives in towns and cities, and urban areas are expanding faster than any other land-use type. In response to this phenomenon, two opposing arguments have emerged: whether cities should ‘sprawl’ into the wider countryside, or ‘densify’ through the development of existing urban greenspace. However, these greenspaces are increasingly recognized as being central to the amelioration of urban living conditions, supporting biodiversity conservation and ecosystem service provision. Taking the highly urbanized region of England as a case study, we use data from a variety of sources to investigate the impact of national-level planning policy on temporal patterns in the extent of greenspace in cities. Between 1991 and 2006, greenspace showed a net increase in all but one of 13 cities. However, the majority of this gain occurred prior to 2001, and greenspace has subsequently declined in nine cities. Such a dramatic shift in land use coincides with policy reforms in 2000, which favoured densification. Here, we illustrate the dynamic and policy-responsive nature of urban land use, thereby highlighting the need for a detailed investigation of the trade-offs associated with different mechanisms of urban densification to optimize and secure the diverse benefits associated with greenspaces.
urbanization; ecosystem services; human population density; urban densification; urban ecology; urban greenspace
Based on the recently established role for the master coregulator MTA1 and MTA1-containing nuclear remodeling complexes, in oncogenesis and inflammation, we explored the links between parasitism by the carcinogenic liver fluke Opisthorchis viverrini and this coregulator using both an Mta1−/− mouse model of infection and a tissue microarray of liver fluke induced human cholangiocarcinomas. Intense foci of inflammation and periductal fibrosis in the liver and kidney of wild type, Mta1+/+ mice were evident at 23 days post-infection with O. viverrini. In contrast, little inflammatory response was observed in the same organs of infected Mta1−/− mice. Livers of infected Mta1+/+ mice revealed strong upregulation of fibrosis-associated markers such as cytokeratins 18, 19, and annexin-2, as determined by both by immunostaining and by reverse transcription PCR compared to infected Mta1−/− mice. CD4 expression was upregulated by infection in the liver of both experimental groups; however, its levels were several folds higher in the Mta1+/+ mice than in infected Mta1−/− mice. Mta1−/− infected mice also exhibited significantly higher systemic and hepatic levels of host cytokines such as IL-12p70, IL-10 and IFN-γ compared to the levels of these cytokines in the Mta1+/+ mice, suggesting an essential role of MTA1 in the cross-regulation of the Th1 and Th2 responses, presumably due to chromatin remodeling of the target chromatin genes. Immunohistochemical analysis of ~300 liver tissue cores from confirmed cases of O. viverrini induced cholangiocarcinoma showed that MTA1 expression was elevated in more than 80% of the specimens. These findings suggest that MTA1 status plays an important role in conferring an optimal cytokine response in mice following infection with O. viverrini and is a major player in parasite-induced cholangiocarcinoma in humans.
Liver and bile duct cancer; Liver Fluke; Tumor Biology; Cancer-causing pathogens; Metastasis associated gene-1 product; host factor for pathogens
Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and the Lao People’s Democratic Republic (Lao PDR; Laos). Currently, more than 600 million people are at risk of infection with these fish-borne trematodes and/or their close relatives. Opisthorchiasis has been studied extensively in Thailand, where about 8 million people are infected with the liver fluke. Here we review the pathogenesis, control and re-emergence of O. viverrini infection, in particular in Thailand and, to a lesser extent in Lao PDR given the contiguous geographical range of O. viverrini through these two regions. We also review the association of O. viverrini infection and cholangiocarcinoma, bile duct cancer, and highlight new findings on pathogenesis of liver fluke induced cholangiocarcinogenesis. Last, we comment on national control strategies in Thailand for the control of O. viverrini infection aimed at reduction in the prevalence of O. viverrini-associated liver cancer in the longer term.
Opisthorchiasis; Opisthorchis; Clonorchis; cholangiocarcinoma; liver fluke; liver cancer; food borne trematodiases; anthelmintic; praziquantel; hepatobiliary disease
Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.
Parasitic roundworms sicken and debilitate over one billion people, most of whom subsist on less than two US dollars per day. There are no vaccines and few drugs available to treat and prevent these infections. Basic research leading to new therapies has been hampered because we lack methods to study gene function in parasitic roundworms. One such method is transgenesis, a process by which gene function is inferred by studying the effects of transferring native or altered copies of genes into subject organisms. Our laboratory has developed a system for transferring synthetic genes into parasitic roundworms of the genus Strongyloides and for obtaining temporary expression of these “transgenes”. Until now, however, we have been unable to propagate these transgenic parasites in the laboratory. This paper describes a new technique that allows us to establish and maintain self-perpetuating lines of transgenic parasites for study. This represents a fundamental advance in the methodology for studying gene function in parasitic roundworms and should greatly facilitate the discovery of new therapies.
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.
Sequence data from this study have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/embl) under accession number ERP000379.
Schistosomes, or blood flukes, are responsible for the major neglected tropical disease called schistosomiasis, which afflicts over 200 million people in impoverished regions of the developing world. The genome sequence of these parasites has been decoded. Integration sites of retroviral transgenes into the chromosomes of schistosomes were investigated by high-throughput sequencing. Transgene integrations were mapped to the genome sequence of Schistosoma mansoni. Integrations were distributed apparently randomly across each of the eight chromosomes, including the seven autosomes and the sex chromosomes Z and W. Integration events of transgenes were characterized in chromosomes of cercariae that were progeny of schistosome eggs infected with pseudotyped virions. Also, transgenic cercariae were employed to infect mice and transgenes were detected in the F1 eggs. Together these findings confirmed vertical transmission of transgenes through the schistosome germline, through both the asexual and the sexual reproductive phases of the developmental cycle. Moreover, germline-transmitted retroviral transgenes encoding drug resistance to the aminoglycoside antibiotics allowed schistosomes to survive toxic concentrations of the antibiotic G418. These findings represent the first reports of wide-scale insertional mutagenesis of schistosome chromosomes and vertical transmission of a transgene through the schistosome germline.