Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini.

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 (ΔOv-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 ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-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 ΔOv-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.

Natural-Product-Based Solutions for Tropical Infectious Diseases.

About half of the world's population and 80% of the world's biodiversity can be found in the tropics. Many diseases are specific to the tropics, with at least 41 diseases caused by endemic bacteria, viruses, parasites, and fungi. Such diseases are of increasing concern, as the geographic range of tropical diseases is expanding due to climate change, urbanization, change in agricultural practices, deforestation, and loss of biodiversity. While traditional medicines have been used for centuries in the treatment of tropical diseases, the active natural compounds within these medicines remain largely unknown. In this review, we describe infectious diseases specific to the tropics, including their causative pathogens, modes of transmission, recent major outbreaks, and geographic locations. We further review current treatments for these tropical diseases, carefully consider the biodiscovery potential of the tropical biome, and discuss a range of technologies being used for drug development from natural resources. We provide a list of natural products with antimicrobial activity, detailing the source organisms and their effectiveness as treatment. We discuss how technological advancements, such as next-generation sequencing, are driving high-throughput natural product screening pipelines to identify compounds with therapeutic properties. This review demonstrates the impact natural products from the vast tropical biome have in the treatment of tropical infectious diseases and how high-throughput technical capacity will accelerate this discovery process.

Newly Discovered Peptides from the Coral Heliofungia actiniformis Show Structural and Functional Diversity.

Scleractinian corals are crucially important to the health of some of the world's most biodiverse, productive, and economically important marine habitats. Despite this importance, analysis of coral peptidomes is still in its infancy. Here we show that the tentacle extract from the stony coral Heliofungia actiniformis is rich in peptides with diverse and novel structures. We have characterized the sequences and three-dimensional structures of four new peptides, three of which have no known homologues. We show that a 2 kDa peptide, Hact-2, promotes significant cell proliferation on human cells and speculate this peptide may be involved in the remarkable regenerative capacity of corals. We found a 3 kDa peptide, Hact-3, encoded within a fascin-like domain, and homologues of Hact-3 are present in the genomes of other coral species. Two additional peptides, Hact-4 and Hact-SCRiP1, with limited sequence similarity, both contain a beta-defensin-like fold and highlight a structural link with the small cysteine-rich proteins (SCRiP) family of proteins found predominantly in corals. Our results provide a first glimpse into the remarkable and unexplored structural diversity of coral peptides, providing insight into their diversity and putative functions and, given the ancient lineage of corals, potential insight into the evolution of structural motifs.

Orally Administered Bacillus Spores Expressing an Extracellular Vesicle-Derived Tetraspanin Protect Hamsters Against Challenge Infection With Carcinogenic Human Liver Fluke.

BACKGROUND: The human liver fluke Opisthorchis viverrini is a food-borne trematode that causes hepatobiliary disease in humans throughout Southeast Asia. People become infected by consuming raw or undercooked fish containing metacercariae. Development of a vaccine to prevent or minimize pathology would decrease the risk of severe morbidity, including the development of bile duct cancer. METHODS: We produced an oral vaccine based on recombinant Bacillus subtilis spores expressing the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (Ov-TSP-2), a protein that is abundant on the surface of O. viverrini secreted extracellular vesicles (EVs). Recombinant spores expressing Ov-TSP-2-LEL were orally administered to hamsters prior to challenge infection with O. viverrini metacercariae. RESULTS: Vaccinated hamsters generated serum IgG as well as bile IgG and IgA responses to Ov-TSP-2-LEL, and serum IgG from vaccinated hamsters blocked the uptake of fluke EVs by a human bile duct epithelial cell line. Vaccinated hamsters had 56% reductions in both adult flukes and fecal eggs compared to the control group. CONCLUSIONS: These findings indicate that oral vaccination of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL is efficacious at reducing infection intensity and could form the basis of a vaccine for control of carcinogenic liver fluke infection in humans.

Developmental Sensitivity in Schistosoma mansoni to Puromycin To Establish Drug Selection of Transgenic Schistosomes.

Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC50) for schistosomula was 19 µg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development of in vitro-laid eggs (LC50, 68 ng/ml) but was less effective against liver eggs (LC50, 387 µg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LC50s after 48 h of 4.9 and 17.3 µg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.

Structural Characterisation of Predicted Helical Regions in the Chironex fleckeri CfTX-1 Toxin.

The Australian jellyfish Chironex fleckeri, belongs to a family of cubozoan jellyfish known for their potent venoms. CfTX-1 and -2 are two highly abundant toxins in the venom, but there is no structural data available for these proteins. Structural information on toxins is integral to the understanding of the mechanism of these toxins and the development of an effective treatment. Two regions of CfTX-1 have been predicted to have helical structures that are involved with the mechanism of action. Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution. This lack of structure concurs with structural disorder predicted for this region of the protein using the Protein DisOrder prediction System PrDOS. Conversely, a peptide corresponding to a predicted transmembrane region is very hydrophobic, insoluble in aqueous solution and predicted to be structured by PrDOS. In the presence of SDS-micelles both peptides have well-defined helical structures showing that a membrane mimicking environment stabilizes the structures of both peptides and supports the prediction of the transmembrane region in CfTX-1. This is the first study to experimentally analyze the structure of regions of a C. fleckeri protein.

Defined Small Molecules Produced by Himalayan Medicinal Plants Display Immunomodulatory Properties.

Plant-derived compounds that modulate the immune responses are emerging as frontline treatment agents for cancer, infectious diseases and autoimmunity. Herein we have isolated 40 phytochemicals from five Bhutanese Sowa Rigpa medicinal plants-Aconitum laciniatum, Ajania nubegina, Corydalis crispa, Corydalis dubia and Pleurospermum amabile-and tested 14 purified compounds for their immunomodulatory properties using a murine dendritic cell (DC) line, and cytotoxicity against a human cholangiocyte cell line using xCELLigence real time cell monitoring. These compounds were: pseudaconitine, 14-veratryolpseudaconitine, 14-O-acetylneoline, linalool oxide acetate, (E)-spiroether, luteolin, luteolin-7-O-ß-d-glucopyranoside, protopine, ochrobirine, scoulerine, capnoidine, isomyristicin, bergapten, and isoimperatorin. Of the 14 compounds tested here, scoulerine had adjuvant-like properties and strongly upregulated MHC-I gene and protein expression whereas bergapten displayed immunosuppressive properties and strongly down-regulated gene and protein expression of MHC-I and other co-stimulatory molecules. Both scoulerine and bergapten showed low cytotoxicity against normal healthy cells that were consistent with their immunoregulatory properties. These findings highlight the breadth of immunomodulatory properties of defined compounds from Bhutanese medicinal plants and show that some of these compounds exert their mechanisms of action by modulating DC activity.

Carcinogenic Parasite Secretes Growth Factor That Accelerates Wound Healing and Potentially Promotes Neoplasia.

Infection with the human liver fluke Opisthorchis viverrini induces cancer of the bile ducts, cholangiocarcinoma (CCA). Injury from feeding activities of this parasite within the human biliary tree causes extensive lesions, wounds that undergo protracted cycles of healing, and re-injury over years of chronic infection. We show that O. viverrini secreted proteins accelerated wound resolution in human cholangiocytes, an outcome that was compromised following silencing of expression of the fluke-derived gene encoding the granulin-like growth factor, Ov-GRN-1. Recombinant Ov-GRN-1 induced angiogenesis and accelerated mouse wound healing. Ov-GRN-1 was internalized by human cholangiocytes and induced gene and protein expression changes associated with wound healing and cancer pathways. Given the notable but seemingly paradoxical properties of liver fluke granulin in promoting not only wound healing but also a carcinogenic microenvironment, Ov-GRN-1 likely holds marked potential as a therapeutic wound-healing agent and as a vaccine against an infection-induced cancer of major public health significance in the developing world.

Conotoxin Φ-MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti-Apoptotic Activity.

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two ß-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85 µm) while inhibiting apoptosis (EC50 2.2 µm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors.

Transcriptional responses of in vivo praziquantel exposure in schistosomes identifies a functional role for calcium signalling pathway member CamKII.

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 "Ca(2+) 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 Ca(2+)-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 Ca(2+) 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.

Suppression of Ov-grn-1 encoding granulin of Opisthorchis viverrini inhibits proliferation of biliary epithelial cells.

Multistep processes likely underlie cholangiocarcinogenesis induced by chronic infection with the fish-borne liver fluke, Opisthorchis viverrini. One process appears to be cellular proliferation of the host bile duct epithelia driven by excretory-secretory (ES) products of this pathogen. Specifically, the secreted growth factor Ov-GRN-1, a liver fluke granulin, is a prominent component of ES and a known driver of hyper-proliferation of cultured human and mouse cells in vitro. We show potent hyper-proliferation of human cholangiocytes induced by low nanomolar levels of recombinant Ov-GRN-1 and similar growth produced by low microgram concentrations of ES products and soluble lysates of the adult worm. To further explore the influence of Ov-GRN-1 on the flukes and the host cells, expression of Ov-grn-1 was repressed using RNA interference. Expression of Ov-grn-1 was suppressed by 95% by day 3 and by ~100% by day 7. Co-culture of Ov-grn-1 suppressed flukes with human cholangiocyte (H-69) or human cholangiocarcinoma (KKU-M214) cell lines retarded cell hyper-proliferation by 25% and 92%, respectively. Intriguingly, flukes in which expression of Ov-grn-1 was repressed were less viable in culture, suggesting that Ov-GRN-1 is an essential growth factor for survival of the adult stage of O. viverrini, at least in vitro. To summarize, specific knock down of Ov-grn-1 reduced in vitro survival and capacity of ES products to drive host cell proliferation. These findings may help to contribute to a deeper understanding of liver fluke induced cholangiocarcinogenesis.

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.

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

The liver fluke Opisthorchis viverrini (Poirier, 1886) (Digenea) secretes extracellular vesicles (EVs) bearing CD63-like tetraspanins on their surface. Fluke EVs are actively internalised 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 hours (P < 0.05) and 48 hours (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-2 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.

Peptide derived from progranulin of the carcinogenic liver fluke, Opisthorchis viverrini stimulates cell hyperproliferation and proinflammatory cytokine production.

Purpose: Progranulin (PGRN) is a secreted glycoprotein growth factor with roles in wound healing, inflammation, angiogenesis and malignancy. An orthologue of the gene encoding human PGRN was identified in the carcinogenic liver fluke Opisthorchis viverrini. Methods: Sequence structure, general characteristics and possible function of O. viverrini PGRN was analyzed using bioinformatics. Expression profiles were investigated with quantitative RT-PCR, western blot and immunolocalization. A specific peptide of Ov-PGRN was used to investigate a role for this molecule in pathogenesis. Results: The structure of the gene coding for O. viverrini PGRN was 36,463 bp in length, and comprised of 13 exons, 12 introns, and a promoter sequence. The Ov-pgrn mRNA is 2,768 bp in length and encodes an 846 amino acids with a predicted molecular mass of 91.61 kDa. Ov-PGRN exhibited one half and seven complete granulin domains. Phylogenetic analysis revealed that Ov-PGRN formed its closest relationship with PGRN of liver flukes in the Opisthorchiidae. Transcripts of Ov-pgrn were detected in several developmental stages, with highest expression in the metacercaria, indicating that Ov-PGRN may participate as a growth factor in the early development of O. viverrini. Western blot analysis revealed the presence of detected Ov-PGRN in both soluble somatic or excretory/secretory products, and immunolocalization indicated high levels of expression in the tegument and parenchyma of the adult fluke. Co-culture of a human cholangiocyte cell line and a peptide fragment of Ov-PGRN stimulated proliferation of cholangiocytes and upregulation of expression of the cytokines IL6 and IL8. Conclusion: Ov-PGRN is expressed throughout the life cycle of liver fluke, and likely plays a key role in development and growth.

A granulin-like growth factor secreted by the carcinogenic liver fluke, Opisthorchis viverrini, promotes proliferation of host cells.

The human liver fluke, Opisthorchis viverrini, infects millions of people throughout south-east Asia and is a major cause of cholangiocarcinoma, or cancer of the bile ducts. The mechanisms by which chronic infection with O. viverrini results in cholangiocarcinogenesis are multi-factorial, but one such mechanism is the secretion of parasite proteins with mitogenic properties into the bile ducts, driving cell proliferation and creating a tumorigenic environment. Using a proteomic approach, we identified a homologue of human granulin, a potent growth factor involved in cell proliferation and wound healing, in the excretory/secretory (ES) products of the parasite. O. viverrini granulin, termed Ov-GRN-1, was expressed in most parasite tissues, particularly the gut and tegument. Furthermore, Ov-GRN-1 was detected in situ on the surface of biliary epithelial cells of hamsters experimentally infected with O. viverrini. Recombinant Ov-GRN-1 was expressed in E. coli and refolded from inclusion bodies. Refolded protein stimulated proliferation of murine fibroblasts at nanomolar concentrations, and proliferation was inhibited by the MAPK kinase inhibitor, U0126. Antibodies raised to recombinant Ov-GRN-1 inhibited the ability of O. viverrini ES products to induce proliferation of murine fibroblasts and a human cholangiocarcinoma cell line in vitro, indicating that Ov-GRN-1 is the major growth factor present in O. viverrini ES products. This is the first report of a secreted growth factor from a parasitic worm that induces proliferation of host cells, and supports a role for this fluke protein in establishment of a tumorigenic environment that may ultimately manifest as cholangiocarcinoma.

Expression, refolding and purification of Ov-GRN-1, a granulin-like growth factor from the carcinogenic liver fluke, that causes proliferation of mammalian host cells.

Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver fluke of humans, Opisthorchis viverrini, and showed that recombinant Ov-GRN-1 expressed and refolded from Escherichia coli caused proliferation of mammalian cell lines at nanomolar concentrations. We now report on an optimized method to express and purify monomeric Ov-GRN-1 in E. coli using a straightforward and scalable purification and refolding process. Purified monomeric protein caused proliferation at nanomolar concentrations of cancerous and non-cancerous cell lines derived from human bile duct tissue. The expression and purification method we describe herein will serve as a backbone upon which to develop expression and purification processes for recombinant GRNs from other organisms, accelerating research on this intriguing family of proteins.

The secreted and surface proteomes of the adult stage of the carcinogenic human liver fluke Opisthorchis viverrini.

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The excretory/secretory included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument-host interface since more than ten surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.

Folding of Truncated Granulin Peptides.

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a "mini-granulin" structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

Liver fluke granulin promotes 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.

IPSE, an abundant egg-secreted protein of the carcinogenic helminth Schistosoma haematobium, promotes proliferation of bladder cancer cells and angiogenesis.

BACKGROUND: Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted "infiltrin" protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE's effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium. SUMMARY: Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium.