Biology subjects

Brindley, P. J.

Publications and source records attributed to Brindley, P. J..

16 recordsLinked to original sources

bioRxiv2025

Fluke-borne viruses are a risk factor for diseases caused by carcinogenic trematodes

Three trematode parasites, Schistosoma haematobium, Opisthorchis viverrini, and Clonorchis sinensis are recognized group 1 carcinogens for their roles in squamous cell carcinoma of the urinary bladder and bile duct cancer (cholangiocarcinoma). We aimed to assess the presence of fluke viruses and their association with malignancy in fluke infections. We showed that all three carcinogenic trematodes harbored related viruses. Focusing on O. viverrini, we observed virus persistence at discrete life stages and viral tropism for the fluke tegument, and we provided molecular and serological evidence of parasitized hosts exposure to viral RNA and proteins. Serological analyses confirmed seroconversion of residents in endemic sites and revealed that antibody titers and avidity against fluke-borne viruses increase in individuals with hepatobiliary diseases. Random forest models discriminated efficiently between liver-fluke-infected participants without disease and those with periductal fibrosis or cholangiocarcinoma. Thus, we show that fluke-borne viruses are risk factors and diagnostic targets for fluke-associated cancer.

microbiology↗

bioRxiv2023

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT A SCHISTOSOMA MANSONI GENOME SAFE HARBOR

Recently, we reported programmed Cas9 mediated insertion of a reporter gene into a gene safe harbor site, GSH1, of Schistosoma mansoni via homology-directed repair (HDR) using overlapping guide RNAs. Here, we report efficient and precise CRISPR/Cas12a-mediated homology directed insertion (knockin, KI) of a 5 C6-PEG10-modified double-stranded transgene bearing microhomology arms, 50 nt in length, at GSH1. At the outset, we undertook bioinformatic and computational analysis following by experimental verification of the regulatory activity of endogenous schistosome ubiquitin (SmUbi) promoter and terminator, to drive strong reporter gene expression. Green fluorescent protein activity driven by SmUbi followed electroporation-mediated transfection of schistosome eggs. HDR induced by RNA-guided CRISPR/Cas12a, which releases overhanging DNA strands of 18-24, delivered more efficient KI than CRISPR/Cas9. In this non-model pathogen, programmed KI facilitated precise chromosomal integration of the reporter-gene with at GSH1. The approach advances schistosome transgenesis field and may also advance functional genomics and transfection methods in related parasitic and non-parasitic helminths, which hitherto lack these tools. Author summaryGenome editing (CRISPR) technology is revolutionizing advances in biology, medicine, and agriculture. Transgenesis approaches are integral in diverse applications including gene therapy, biotherapeutics, deciphering host-pathogen interactions, and enhancements in agricultural production. Parasitic worms that are responsible for infectious diseases including neglected tropical diseases (NTDs), which cause substantial morbidity and mortality. NTDs mainly occur in the Global South, and they are responsible for a disease burden that exceeds that caused by malaria and tuberculosis. Infections with parasitic helminths also are responsible for immense economic burden in the agriculture. Tools for functional genomics in parasitic helminths are limited. Access to CRISPR-based approaches can be expected to hasten development of drug and/or vaccine targets for these diseases. Here, we focused on the helminth Schistosoma mansoni, a water borne parasite of humans, and which is endemic in Africa, and northeastern South America. To advance the state of the art in laboratory techniques currently used to study the biology and pathogenesis of this and related pathogens, we evaluated a spectrum of technological approaches aimed at improved current lab practice in this field. The findings demonstrated that specific technical and chemical modifications, including deploying a DNA cutting enzyme termed Cas12a along with a transgene with chemically modified short flanking sequences (homology arms) provided improved gene editing efficiency for this schistosome.

molecular biology↗

bioRxiv2023

Tetraspanins from Opisthorchis viverrini stimulate cholangiocyte migration and inflammatory cytokine production.

The liver fluke Opsithorchis viverrini secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalized by host cholangiocytes in the bile ducts, where they drive pathology and promote neoplasia through induction of cellular proliferation and secretion of inflammatory cytokines. We investigated the effects of tetraspanins of the CD63 superfamily by co-culturing recombinant forms of the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (rLEL-Ov-TSP-2) and tetraspanin-3 (rLEL-Ov-TSP-3) with non-cancerous human bile duct (H69) and cholangiocarcinoma (CCA, M213) cell lines. The results showed that cell lines co-cultured with excretory/secretory products from adult O. viverrini (Ov-ES) underwent significantly increased cell proliferation at 48 hours but not 24 hours compared to untreated control cells (P<0.05), whereas rLEL-Ov-TSP-3 co-culture resulted in significantly increased cell proliferation at both 24 hr (P<0.05) and 48 hr (P<0.01) time points. In like fashion, H69 cholangiocytes co-cultured with both Ov-ES and rLEL-Ov-TSP-3 underwent significantly elevated Il-6 and Il-8 gene expression for at least one of the time points assessed. Finally, both rLEL-Ov-TSP-and rLEL-Ov-TSP-3 significantly enhanced migration of both M213 and H69 cell lines. These findings indicated that O. viverrini CD63 family tetraspanins can promote a cancerous microenvironment by enhancing innate immune responses and migration of biliary epithelial cells.

cell biology↗

bioRxiv2022

Targeted insertion of reporter transgene into a gene safe harbor in human blood fluke, Schistosoma mansoni

The identification and characterization of genomic safe harbor sites (GSH) aims to facilitate consistent transgene activity without disruption to the host cell genome. We combined genome annotation and chromatin structure analysis by computational approach to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. Introduction of a transgene into the egg of the parasite was accomplished using CRISPR/Cas-assisted homology-directed repair and overlapping guide RNAs. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. These outcomes advance functional genomics for schistosomes by providing a tractable path towards transgenic helminths using homology directed repair-catalyzed transgene insertion. This approach should be adaptable to helminths generally. MotivationFunctional genomics methods are needed in the arena of the Neglected Tropical Diseases, especially for helminth parasites, to facilitate basic and transformational studies to improve global public health. Gain-of-function phenotypes would be valuable in this context. Hence, the motivation for this investigation was to identify a genome safe harbor (GSH) site in the chromosomes of the human blood fluke, Schistosoma mansoni and, in a pilot approach, and to develop methods for transgene insertion and methods to characterize transgene performance following homology directed insertion at the schistosome GSH. The progress reported here can be expected to advance functional genomics for species of the Platyhelminthes - including model, free living species of planarians, and should be adaptable to helminths generally.

molecular biology↗

bioRxiv2022

Differential excretory/secretory proteome of the adult female and male stages of the human blood fluke, Schistosoma mansoni

Intricate molecular communication between the schistosome (flatworms) and its mammalian host, as well as between paired male and female schistosomes has shaped the secreted proteome of these flatworms. Whereas the schistosome egg is responsible for the disease manifestations of chronic schistosomiasis, the long lived, adult female and male stages also release mediators that facilitate their long-lived intra-vascular existence in a hostile niche where they are bathed in immune cells and effector molecules. However, despite their importance, no studies have focused on analysing the excretory/secretory products (ESPs) from adult schistosomes. Herein, ESPs from cultured Schistosoma mansoni male or female adult worms were identified, quantified, compared and contrasted using a label-free proteomic approach. Approximately 1,000 proteins were identified, from which almost 800 could be finally quantified. Considering the proteins uniquely identified and proteins with a significantly regulated expression pattern in male or female flukes, a total of 370 and 140 proteins were more abundantly secreted by males and females, respectively. Using functional analysis networks showing the gene ontology terms and KEGG pathways with the highest significance, we observed that male schistosomes secrete proteins related to carbohydrate metabolism, cytoskeletal organisation more abundantly than females, while female worms secreted more hydrolases and proteins involved in cellular homeostasis than males. This analysis doubles the number of previously reported ESPs from S. mansoni, contributing to a better understanding of the host-parasite dynamic interactions. Furthermore, these findings expand potential vaccine and diagnostic candidates for this neglected tropical disease pathogen, which will enable deeper understanding of the molecular communication critical to parasitism.

microbiology↗

bioRxiv2021

Programmed knockout mutation of liver fluke granulin, Ov-grn-1, attenuates virulence and impedes malignant transformation during chronic opisthorchiasis

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes ({Delta}Ov-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, however less hepatobiliary tract disease manifested during chronic infection with{Delta} Ov-grn-1 worms in comparison to hamsters infected with control gene-edited and non-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with{Delta} Ov-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis. Author summaryInfection with the human liver flukes, Opisthorchis viverrini, O. felineus and Clonorchis sinensis remains a public health concern in regions where these parasites are endemic. O. viverrini is endemic in the Mekong River drainage countries of including Thailand and the Lao Peoples Democratic Republic. Infection follows the consumption of undercooked freshwater fish harboring the parasite. Liver fluke infection, opisthorchiasis, is associated with diseases of the liver and bile ducts including cancer of the biliary tract, cholangiocarcinoma, a cancer with a poor prognosis. This report characterizes for the first time experimental infection with gene-edited O. viverrini liver flukes during concurrent exposure to nitrosamine in a rodent model of liver fluke infection-associated cancer. Cancer development was slowed in hamsters infected with the parasite following CRISPR-based knock-out mutation and loss of a parasite gene known to stimulate growth of cells lining the bile ducts. These findings describe a new model for investigation of risk factors for infection-associated cholangiocarcinoma and to assess efficacy of anti-infection/anti-cancer vaccines.

cancer biology↗

bioRxiv2021

RNA-guided AsCas12a- and SpCas9-catalyzed knockout and homology directed repair of the omega-1 locus of the human blood fluke, Schistosoma mansoni

We compared the efficiency of gene knockout (KO) and precision of insertion (knock-in, KI) of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with that of SpCas9 from Streptococcus pyogenes, aiming to enhance the functional genomics toolkit for Schistosoma mansoni. Programmed DNA cleavages catalyzed by Cas12a and Cas9 result in staggered and blunt ended strand breaks, respectively. TTTV, the optimal protospacer adjacent motif for AsCas12a would occur frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key enzyme termed omega-1 that is secreted by the schistosome egg. AsCas12a was more efficient than SpCas9 for gene knockout of omega-1 as determined by tracking of indels by decomposition (P < 0.001). Resulting from CRISPREsso2 analysis, most mutations were deletions; SpCas9 induced short deletions of 3 nt in length whereas AsCas12a induced deletions of 2 to 26 nt. Knockout efficiency of both nucleases markedly increased in the presence of short, single stranded oligodeoxynucleotide (ssODN) donor templates. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands of the targeted gene were tested, resulting in KO efficiencies of 15.67, 28.71 and 21.43% in the SpCas9 plus donor ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans cleavage activity against the ssODNs by activated AsCas12a was not apparent in vitro. Programmed SpCas9 editing led to more precise transgene insertion than AsCas12a, with KI efficiencies of 17.07% for the KI_SpCas9 group, 14.58% for KI_AsCas12a-NT-ssODN and 12.37% for KI_AsCas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases. These findings revealed that AsCas12a and SpCas9 both provide tractable routes for RNA-guided programmed mutation of the genome of the schistosome egg.

molecular biology↗

bioRxiv2021

The transcriptome of Schistosoma mansoni developing eggs reveals key mediators in pathogenesis and life cycle propagation.

Schistosomiasis, the most important helminthic disease of humanity, is caused by infection with parasitic flatworms of the genus Schistosoma. The disease is driven by the eggs laid by the parasites and becoming trapped in host tissues, followed by inflammation and granuloma formation. Despite abundant transcriptome data for most developmental stages of the three main human-infective schistosome species, i.e. Schistosoma mansoni, S. japonicum and S. haematobium, the transcriptomic profiles of developing eggs remain largely unexplored. In this study, we performed RNAseq of S. mansoni eggs laid in vitro during early and late embryogenesis (days 1-3 and 3-6 post-oviposition, respectively). Analysis of the transcriptomes identified hundreds of up-regulated genes during the later stage, including venom allergen-like (VAL) proteins, well-established host immunomodulators, and genes involved in organogenesis of the miracidium larva. In addition, the transcriptomes of the in vitro laid eggs were compared with existing publicly available RNA-seq dataset from S. mansoni eggs collected from the livers of murine hosts. Analysis of enriched GO terms and pathway annotations revealed cell division and protein synthesis processes associated with early embryogenesis, whereas cellular metabolic processes, microtubule-based movement, and microtubule cytoskeleton organization were found enriched in the later developmental time point. This is the first transcriptomic analysis of S. mansoni embryonic development, and will facilitate our understanding of infection pathogenesis, miracidia development and life cycle progression of schistosomes.

developmental biology↗

bioRxiv2021

PIWI silencing mechanism involving the retrotransposon nimbus orchestrates resistance to infection with Schistosoma mansoni in the snail vector, Biomphalaria glabrata

BackgroundSchistosomiasis remains widespread in many regions despite efforts at its elimination. By examining changes in the transcriptome at the host-pathogen interface in the snail Biomphalaria glabrata and the blood fluke Schistosoma mansoni, we previously demonstrated that an early stress response in juvenile snails, manifested by induction of heat shock protein 70 (Hsp 70) and Hsp 90 and of the reverse transcriptase (RT) domain of the B. glabrata non-LTR-retrotransposon, nimbus, were critical for B. glabrata susceptibility to S. mansoni. Subsequently, juvenile B. glabrata BS90 snails, resistant to S. mansoni at 25{degrees}C become susceptible by the F2 generation when maintained at 32{degrees}C, indicating an epigenetic response. Methodology/Principal FindingsTo better understand this plasticity in susceptibility of the BS90 snail, mRNA sequences were examined from S. mansoni exposed juvenile BS90 snails cultured either at 25{degrees}C (permissive temperature) or 32{degrees}C (non-permissive). Comparative analysis of transcriptomes from snails cultured at the non-permissive and permissive temperatures revealed that whereas stress related transcripts dominated the transcriptome of susceptible BS90 juvenile snails at 32{degrees}C, transcripts encoding proteins with a role in epigenetics, such as PIWI (BgPiwi), chromobox protein homolog 1 (BgCBx1), histone acetyl transferase histone deacetylase (HDAC) and metallotransferase (MT) were highly expressed in those cultured at 25{degrees}C. To further determine a role for BgPiwi in B. glabrata susceptibility to S. mansoni, siRNA corresponding to the BgPiwi encoding transcript was utilized to suppress expression of BgPiwi, rendering the resistant BS90 juvenile snail susceptible to infection at 25{degrees}C. Given transposon silencing activity of PIWI as a facet of its role as guardian of the integrity of the genome, we examined the expression of the nimbus RT encoding transcript at 120 min after infection of resistant BS90 piwi-siRNA treated snails. We observed that nimbus RT was upregulated, indicating that modulation of the transcription of the nimbus RT was associated with susceptibility to S. mansoni in BgPiwi- siRNA treated BS90 snails. Furthermore, treatment of susceptible snails with the RT inhibitor lamivudine, before exposure to S. mansoni, blocked S. mansoni infection concurrent with downregulation of the nimbus RT transcript and upregulation of the BgPiwi encoding transcript in the lamivudine-treated, schistosome-exposed susceptible snails. Conclusions and SignificanceThese findings support a role for the interplay of BgPiwi and nimbus in the epigenetic modulation of plasticity of resistance/susceptibility in the snail-schistosome relationship.

molecular biology↗

bioRxiv2020

Tumor Necrosis Factor and Schistosoma mansoni egg antigen Omega-1 shape distinct aspects of the early egg-induced granulomatous response

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.

microbiology↗

bioRxiv2020

Infection with CagA+ Helicobacter pylori induces epithelial to mesenchymal transition in human cholangiocytes

Recent reports suggest that the East Asian liver fluke, Opisthorchis viverrini, infection with which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the cell of origin of this malignancy. Here, we investigated interactions in vitro among human cholangiocytes, a CagA-positive strain of Helicobacter pylori, and the related bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte induced phenotypic changes including the profusion of thread-like filopodia and a loss of cell-cell contact, in a dose-dependent fashion. In parallel, following exposure to H. pylori, changes were evident in levels of mRNA expression of epithelial to mesenchymal transition (EMT)-encoding factors including snail, slug, vimentin, matrix metalloprotease, zinc finger E-box-binding homeobox, and the cancer stem cell marker CD44. Transcription levels encoding the cell adhesion marker CD24 decreased. Analysis to quantify cellular proliferation, migration and invasion in real time using the xCELLigence approach revealed that exposure to [≥]10 H. pylori stimulated migration and invasion by the cholangiocytes through an extracellular matrix. In addition, 10 bacilli of CagA-positive H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection with H. pylori contributes to the malignant transformation of the biliary epithelium.

molecular biology↗

bioRxiv2020

Diminished adherence of snail hemocytes to schistosome sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor of Biomphalaria glabrata

BackgroundLarval development in an intermediate host gastropod snail of the genus Biomphalaria is an obligatory component of the life cycle of Schistosoma mansoni. Understanding of the mechanism(s) of host defense may hasten the development of tools that block transmission of schistosomiasis. The allograft inflammatory factor 1, AIF, which is evolutionarily conserved and expressed in phagocytes, is a marker of macrophage activation in both mammals and invertebrates. AIF enhances cell proliferation and migration. The embryonic cell line, termed Bge, from Biomphalaria glabrata is a versatile resource for investigation of the snail-schistosome relationship since Bge exhibits a hemocyte-like phenotype. Hemocytes perform central roles in innate and cellular immunity in gastropods and in some cases can kill the parasite. However, the Bge cells do not kill the parasite in vitro. MethodsBge cells were transfected by electroporation with plasmid pCas-BgAIFx4, encoding the Cas9 nuclease and a guide RNA specific for exon 4 of the B. glabrata AIF (BgAIF) gene. Transcript levels for Cas9 and for BgAIF were monitored by quantitative reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. ResultsGene knockout manipulation induced gene-disrupting indels, frequently 1-2 bp insertions and/or 8-30 bp deletions, at the programmed target site; a range from 9 to 17% of the copies of the BgAIF gene in the Bge population of cells were mutated. Transcript levels for BgAIF were reduced by up to 73% (49.5{+/-}20.2% S.D, P [≤] 0.05, n =12). Adherence by BgAIF gene-edited ({Delta}BgAIF) Bge to sporocysts diminished in comparison to wild type cells, although cell morphology did not change. Specifically, as scored by a semi-quantitative cell adherence index (CAI), fewer {Delta}BgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66{+/-}0.10, {Delta}BgAIF, 2.30{+/-}0.22 (P [≤] 0.01). ConclusionThe findings supported the hypothesis that BgAIF plays a role in the adherence of B. glabrata hemocytes to sporocysts during schistosome infection in vitro. This demonstration of the activity of programmed gene editing will enable functional genomics approaches using CRISPR/Cas9 to investigate additional components of the snail-schistosome host-parasite relationship.

molecular biology↗

bioRxiv2019

Exosome-mediated crosstalk stimulated by liver fluke granulin promotes a microenvironment conducive to cholangiocarcinoma

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with fish-borne liver flukes is a major risk factor for cholangiocarcinoma (CCA). The parasite secretes a growth factor termed liver fluke granulin (Ov-GRN-1), a homologue of the human progranulin (huPGRN), which contributes significantly to biliary tract fibrosis and morbidity during infection. Here, exosome-mediated transfer of mRNAs from the human cholangiocyte cell line (H69) was investigated following exposure to Ov-GRN-1, to naive recipient H69 cells. To minimize the influence of endogenous huPGRN, the gene encoding huPGRN was inactivated using CRISPR/Cas9-based gene knock-out. Several huPGRN-depleted cell lines, termed {Delta}huPGRN-H69, were established. These lines exhibited >80% reductions in levels of huPGRN transcripts and protein, both in gene-edited cells and within exosomes released by these cells. Profiles of exosomal RNAs (exRNA) from {Delta}huPGRN-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 mRNAs including MAPK/AKT pathway members were induced by exposure to Ov-GRN-1. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 exposed to Ov-GRN-1. Of these, CCA-associated exRNAs modified the CCA microenvironment in naive recipient cells co-cultured with exosomes from {Delta}huPGRN-H69 exposed to Ov-GRN-1, and induced translation of MAPK phosphorylation related-protein in naive recipient cells comparing 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.

molecular biology↗

bioRxiv2019

Helminth infection-induced carcinogenesis: spectrometric insights from the liver flukes, Opisthorchis and Fasciola

Chronic infections with the flatworm parasites Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium are classified as group 1 biological carcinogens, i.e. definitive causes of cancer. In addition, we reported findings that support the inclusion of Opisthorchis felineus in this list of biological carcinogens. By contrast, infections with close phylogenetic relatives including Fasciola hepatica have not been associated with carcinogenesis. Earlier reports revealed of oxysterol metabolites of Opisthorchis liver fluke origin conjugated with DNA bases, suggesting that the generation of these DNA-adducts may underlie the mutagenicity and carcinogenicity of the infection with these food-borne pathogens. Here we employed liquid chromatography-mass spectrometry (LC-MS/MS) to investigate, compare and contrast spectrograms of soluble extracts from F. hepatica adult worms from bile ducts of cattle with those from O. viverrini and O. felineus from experimentally-infected hamsters. F. hepatica displayed a complex spectrophotometric profile. F. hepatica and Opisthorchis spp. shared several common compounds including oxysterol-like metabolites, bile acids and DNA-adducts, but the spectrometric profiles of these Opisthorchis species included far fewer compounds than F. hepatica. These findings support the postulate that oxysterol-like metabolites of parasite origin can initiate carcinogenesis and they point to a molecular basis for the inconsistencies among major groups of liver flukes concerning infection-induced malignancy.\n\nAuthor SummarySeveral species of trematodes are parasites of the human hepatobiliary tract. Infection with two of these flukes, Clonorchis sinsensis and Opisthorchis viverrini, fresh water fish-borne parasites that occur in East Asia is classified as group 1 carcinogens by the International Agency for Research on Cancer (IARC), i.e. definitive causes of cancer in humans. By contrast, infection with a different liver fluke, Fasciola hepatica, does not lead to malignant transformation of the biliary tract. Given the close phylogeny of all three parasites, this difference in carcinogenicity is intriguing and, if explained, likely of value in novel therapeutic approaches. The importance of the current findings is informative because they present a mass spectrometric analysis and catalog of the similarities and differences between fluke of the genus Opisthorchis and F. hepatica, potentially identifying carcinogenic metabolites of liver fluke origin. These metabolites can be expected to provide deeper understanding of helminth infection induced malignancy.

cancer biology↗

bioRxiv2018

Programmed mutation of liver fluke granulin using CRISPR/Cas9 attenuates virulence of infection-induced hepatobiliary morbidity

Infections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

molecular biology↗

bioRxiv2018

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 ({omega}1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of {omega}1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5-and 3-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within {omega}1. CRISPResso analysis of amplicons spanning the DSB revealed [~]4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding {omega}1 were reduced >80% and lysates of {omega}1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the {omega}1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of {omega}1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding {omega}1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of {omega}1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of {omega}1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

molecular biology↗