Small extracellular vesicles but not microvesicles from Opisthorchis viverrini promote cell proliferation in human cholangiocytes.

Chronic infection with O. viverrini has been linked to the development of cholangiocarcinoma (CCA), which is a major public health burden in the Lower Mekong River Basin countries, including Thailand, Lao PDR, Vietnam and Cambodia. Despite its importance, the exact mechanisms by which O. viverrini promotes CCA are largely unknown. In this study, we characterized different extracellular vesicle populations released by O. viverrini (OvEVs) using proteomic and transcriptomic analyses and investigated their potential role in host-parasite interactions. While 120k OvEVs promoted cell proliferation in H69 cells at different concentrations, 15k OvEVs did not produce any effect compared to controls. The proteomic analysis of both populations showed differences in their composition that could contribute to this differential effect. Furthermore, the miRNAs present in 120k EVs were analysed and their potential interactions with human host genes was explored by computational target prediction. Different pathways involved in inflammation, immune response and apoptosis were identified as potentially targeted by the miRNAs present in this population of EVs. This is the first study showing specific roles for different EV populations in the pathogenesis of a parasitic helminth, and more importantly, an important advance towards deciphering the mechanisms used in establishment of opisthorchiasis and liver fluke infection-associated malignancy.

Study of the migration of Fasciola hepatica juveniles across the intestinal barrier of the host by quantitative proteomics in an ex vivo model.

Fasciola hepatica is a trematode parasite that infects animals and humans causing fasciolosis, a worldwide-distributed disease responsible for important economic losses and health problems. This disease is of growing public health concern since parasite isolates resistant to the current treatment (triclabendazole) have increasingly been described. F. hepatica infects its vertebrate host after ingestion of the encysted parasite (metacercariae), which are found in the water or attached to plants. Upon ingestion, newly excysted juveniles of F. hepatica (FhNEJ) emerge in the intestinal lumen and cross the intestinal barrier, reach the peritoneum and migrate to the biliary ducts, where adult worms fully develop. Despite the efforts made to develop new therapeutic and preventive tools, to date, protection against F. hepatica obtained in different animal models is far from optimal. Early events of host-FhNEJ interactions are of paramount importance for the infection progress in fasciolosis, especially those occurring at the host-parasite interface. Nevertheless, studies of FhNEJ responses to the changing host environment encountered during migration across host tissues are still scarce. Here, we set-up an ex vivo model coupled with quantitative SWATH-MS proteomics to study early host-parasite interaction events in fasciolosis. After comparing tegument and somatic fractions from control parasites and FhNEJ that managed to cross a mouse intestinal section ex vivo, a set of parasite proteins whose expression was statistically different were found. These included upregulation of cathepsins L3 and L4, proteolytic inhibitor Fh serpin 2, and a number of molecules linked with nutrient uptake and metabolism, including histone H4, H2A and H2B, low density lipoprotein receptor, tetraspanin, fatty acid binding protein a and glutathione-S-transferase. Downregulated proteins in FhNEJ after gut passage were more numerous than the upregulated ones, and included the heath shock proteins HSP90 and alpha crystallin, amongst others. This study brings new insights into early host-parasite interactions in fasciolosis and sheds light on the proteomic changes in FhNEJ triggered upon excystment and intestinal wall crossing, which could serve to define new targets for the prevention and treatment of this widespread parasitic disease.

Foodborne trematodes: old foes, new kids on the block and research perspectives for control and understanding host-parasite interactions.

Foodborne trematodes (FBTs) have a worldwide distribution (with particular prevalence in south-east Asia) and are believed to infect almost 75 million people, with millions more living at risk of infection. Although mortality due to trematodiasis is low, these infections cause considerable morbidity and some species are associated with the development of cancer in hyperendemic regions. Despite this, FBTs are often side-lined in terms of research funding and have been dubbed neglected tropical diseases by the World Health Organisation. Thus, the aim of this special issue was to provide an update of our understanding of FBT infections, to shine a light on current work in the field and to highlight some research priorities for the future. With contributions from leading researchers, many from endemic regions, we review the major FBT species. In doing so we revisit some old foes, uncover emerging infections and discover how outbreaks are being dealt with as a result of new approaches to parasite control. We also report advances in our understanding of the interactions of FBTs with their mammalian hosts and uncover new interplay between trematodes and host microbiome components. We hope that this article collection will stimulate discussion and further research on the FBTs and help raise them from their neglected status.

Administration of Hookworm Excretory/Secretory Proteins Improves Glucose Tolerance in a Mouse Model of Type 2 Diabetes.

Diabetes is recognised as the world's fastest growing chronic condition globally. Helminth infections have been shown to be associated with a lower prevalence of type 2 diabetes (T2D), in part due to their ability to induce a type 2 immune response. Therefore, to understand the molecular mechanisms that underlie the development of T2D-induced insulin resistance, we treated mice fed on normal or diabetes-promoting diets with excretory/secretory products (ES) from the gastrointestinal helminth Nippostrongylus brasiliensis. We demonstrated that treatment with crude ES products from adult worms (AES) or infective third-stage larvae (L3ES) from N. brasiliensis improved glucose tolerance and attenuated body weight gain in mice fed on a high glycaemic index diet. N. brasiliensis ES administration to mice was associated with a type 2 immune response measured by increased eosinophils and IL-5 in peripheral tissues but not IL-4, and with a decrease in the level of IL-6 in adipose tissue and corresponding increase in IL-6 levels in the liver. Moreover, treatment with AES or L3ES was associated with significant changes in the community composition of the gut microbiota at the phylum and order levels. These data highlight a role for N. brasiliensis ES in modulating the immune response associated with T2D, and suggest that N. brasiliensis ES contain molecules with therapeutic potential for treating metabolic syndrome and T2D.

Silencing of Opisthorchis viverrini Tetraspanin Gene Expression Results in Reduced Secretion of Extracellular Vesicles.

Inter-phylum transfer of molecular information is exquisitely exemplified in the uptake of parasite extracellular vesicles (EVs) by their target mammalian host tissues. The oriental liver fluke, Opisthorchis viverrini is the major cause of bile duct cancer in people in Southeast Asia. A major mechanism by which O. viverrini promotes cancer is through the secretion of excretory/secretory products which contain extracellular vesicles (OvEVs). OvEVs contain microRNAs that are predicted to impact various mammalian cell proliferation pathways, and are internalized by cholangiocytes that line the bile ducts. Upon uptake, OvEVs drive relentless proliferation of cholangiocytes and promote a tumorigenic environment, but the underlying mechanisms of this process are unknown. Moreover, purification and characterization methods for helminth EVs in general are ill defined. We therefore compared different purification methods for OvEVs and characterized the sub-vesicular compartment proteomes. Two CD63-like tetraspanins (Ov-TSP-2 and TSP-3) are abundant on the surface of OvEVs, and could serve as biomarkers for these parasite vesicles. Anti-TSP-2 and -TSP-3 IgG, as well as different endocytosis pathway inhibitors significantly reduced OvEV uptake and subsequent proliferation of cholangiocytes in vitro. Silencing of Ov-tsp-2 and tsp-3 gene expression in adult flukes using RNA interference resulted in substantial reductions in OvEV secretion, and those vesicles that were secreted were deficient in their respective TSP proteins. Our findings shed light on the importance of tetraspanins in fluke EV biogenesis and/or stability, and provide a conceivable mechanism for the efficacy of anti-tetraspanin subunit vaccines against a range of parasitic helminth infections.

ß-Glucan receptors on IL-4 activated macrophages are required for hookworm larvae recognition and trapping.

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for ß-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed ß-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize ß-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.

Characterisation of tetraspanins from Schistosoma haematobium and evaluation of their potential as novel diagnostic markers.

Schistosoma haematobium is the leading cause of urogenital schistosomiasis and it is recognised as a class 1 carcinogen due to the robust association of infection with bladder cancer. In schistosomes, tetraspanins (TSPs) are abundantly present in different parasite proteomes and could be potential diagnostic candidates due to their accessibility to the host immune system. The large extracellular loops of six TSPs from the secretome (including the soluble excretory/secretory products, tegument and extracellular vesicles) of S. haematobium (Sh-TSP-2, Sh-TSP-4, Sh-TSP-5, Sh-TSP-6, Sh-TSP-18 and Sh-TSP-23) were expressed in a bacterial expression system and polyclonal antibodies were raised to the recombinant proteins to confirm the anatomical sites of expression within the parasite. Sh-TSP-2, and Sh-TSP-18 were identified on the tegument, whereas Sh-TSP-4, Sh-TSP-5, Sh-TSP-6 and Sh-TSP-23 were identified both on the tegument and internal tissues of adult parasites. The mRNAs encoding these TSPs were differentially expressed throughout all schistosome developmental stages tested. The potential diagnostic value of three of these Sh-TSPs was assessed using the urine of individuals (stratified by infection intensity) from an endemic area of Zimbabwe. The three Sh-TSPs were the targets of urine IgG responses in all cohorts, including individuals with very low levels of infection (those positive for circulating anodic antigen but negative for eggs by microscopy). This study provides new antigen candidates to immunologically diagnose S. haematobium infection, and the work presented here provides compelling evidence for the use of a biomarker signature to enhance the diagnostic capability of these tetraspanins.

Immunomics-Guided Antigen Discovery for Praziquantel-Induced Vaccination in Urogenital Human Schistosomiasis.

Despite the enormous morbidity attributed to schistosomiasis, there is still no vaccine to combat the disease for the hundreds of millions of infected people. The anthelmintic drug, praziquantel, is the mainstay treatment option, although its molecular mechanism of action remains poorly defined. Praziquantel treatment damages the outermost surface of the parasite, the tegument, liberating surface antigens from dying worms that invoke a robust immune response which in some subjects results in immunologic resistance to reinfection. Herein we term this phenomenon Drug-Induced Vaccination (DIV). To identify the antigenic targets of DIV antibodies in urogenital schistosomiasis, we constructed a recombinant proteome array consisting of approximately 1,000 proteins informed by various secretome datasets including validated proteomes and bioinformatic predictions. Arrays were screened with sera from human subjects treated with praziquantel and shown 18 months later to be either reinfected (chronically infected subjects, CI) or resistant to reinfection (DIV). IgG responses to numerous antigens were significantly elevated in DIV compared to CI subjects, and indeed IgG responses to some antigens were completely undetectable in CI subjects but robustly recognized by DIV subjects. One antigen in particular, a cystatin cysteine protease inhibitor stood out as a unique target of DIV IgG, so recombinant cystatin was produced, and its vaccine efficacy assessed in a heterologous Schistosoma mansoni mouse challenge model. While there was no significant impact of vaccination with adjuvanted cystatin on adult worm numbers, highly significant reductions in liver egg burdens (45-55%, P<0.0001) and intestinal egg burdens (50-54%, P<0.0003) were achieved in mice vaccinated with cystatin in two independent trials. This study has revealed numerous antigens that are targets of DIV antibodies in urogenital schistosomiasis and offer promise as subunit vaccine targets for a drug-linked vaccination approach to controlling schistosomiasis.

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.

Set up of an in vitro model to study early host-parasite interactions between newly excysted juveniles of Fasciola hepatica and host intestinal cells using a quantitative proteomics approach.

Fasciola hepatica is the causative agent of fasciolosis, a parasitic zoonosis of global distribution causing significant economic losses in animal production and a human public health problem in low-income countries. Hosts are infected by ingestion of aquatic plants carrying metacercariae. Once ingested, the juvenile parasites excyst in the small intestine and, after crossing it, they follow a complex migratory route that lead the parasites to their definitive location in the bile ducts. Despite being a critical event in the progression of the infection, the available data on the cross-talk relationships between the parasite and the host at an early stage of the infection are scarce. The objective of the present work is to characterize the proteomic changes occurring in both the parasite and the host, through the development of a novel in vitro model, to shed light on the molecular pathways of communication between the newly excysted juveniles (NEJ) from F. hepatica and the host's intestinal epithelium. For this, in vitro excystation of F. hepatica metacercariae was carried out and NEJ were obtained. Additionally, optimal conditions of growth and expansion of mouse primary small intestinal epithelial cells (MPSIEC) in culture were fine-tuned. Tegumentary and somatic parasite antigens (NEJ-Teg and NEJ-Som), as well as host cell protein lysate (MPSIEC-Lys) were obtained before and after 24 h co-culture of NEJ with MPSIEC. We used an isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy to detect 191 and 62 up-regulated, and 112 and 57 down-regulated proteins in the NEJ-Teg and NEJ-Som extracts, respectively. Similarly, 87 up-regulated and 73 down-regulated proteins in the MPSIEC-Lys extract were identified. Taking into account the biological processes in which these proteins were involved, interesting mechanisms related to parasite development, invasion and evasion, as well as manipulation of the host intestinal epithelial cell adhesion, immunity and apoptosis pathways, among others, could be inferred, taking place at the host-parasite interface. The further understanding of these processes could constitute promising therapeutic targets in the future against fasciolosis.

Partial protection with a chimeric tetraspanin-leucine aminopeptidase subunit vaccine against Opisthorchis viverrini infection in hamsters.

Opisthorchiasis is a serious public health problem in East Asia and Europe. The pathology involves hepatobiliary abnormalities such as cholangitis, choledocholithiasis and tissue fibrosis that can develop into cholangiocarcinoma. Prevention of infection is difficult as multiple social and behavioral factors are involved, thus, progress on a prophylactic vaccine against opisthorchiasis is urgently needed. Opisthorchis viverrini tetraspanin-2 (Ov-TSP-2) was previously described as a potential vaccine candidate conferring partial protection against O. viverrini infections in hamsters. In this study, we generated a recombinant chimeric form of the large extracellular loop of Ov-TSP-2 and O. viverrini leucine aminopeptidase, designated rOv-TSP-2-LAP. Hamsters were vaccinated with 100 and 200 µg of rOv-TSP-2-LAP formulated with alum-CpG adjuvant via intraperitoneal injection and evaluated the level of protection against O. viverrini infection. Our results demonstrated that the number of worms recovered from hamsters vaccinated with either 100 or 200 µg of rOv-TSP-2-LAP were significantly reduced by 27% compared to the adjuvant control group. Furthermore, the average length of worms recovered from animals vaccinated with 200 µg of rOv-TSP-2-LAP was significantly shorter than those from the control adjuvant group. Immunized hamsters showed significantly increased serum levels of anti-rOv-TSP-2 IgG and IgG1 compared to adjuvant control group, suggesting that rOv-TSP-2-LAP vaccination induces a mixed Th1/Th2 immune response in hamsters. Therefore, the development of a suitable vaccine against opisthorchiasis requires further work involving new vaccine technologies to improve immunogenicity and protective efficacy.

Recombinant Opisthorchis viverrini tetraspanin expressed in Pichia pastoris as a potential vaccine candidate for opisthorchiasis.

Opisthorchiasis affects millions of people in Southeast Asia and has been strongly associated with bile duct cancer. Current strategic control approaches such as chemotherapy and health education are not sustainable, and a prophylactic vaccine would be a major advance in the prevention of the disease. Tetraspanins are transmembrane proteins previously described as potential vaccine candidates for other helminth infections and are also found in the membranes of the tegument and extracellular vesicles of O. viverrini. Here, we investigated the potential of a recombinant protein encoding for the large extracellular loop of O. viverrini tetraspanin-2 (rOv-LEL-TSP-2) in a hamster vaccination model. Hamsters were vaccinated with 50 and 100 µg of rOv-LEL-TSP-2 produced from Pichia pastoris yeast combined with alum CpG adjuvant via the intraperitoneal route. The number of worms recovered from hamsters vaccinated with rOv-LEL-TSP-2 was significantly reduced compared to adjuvant control groups. Fecal egg output was also significantly reduced in vaccinated animals, and the average length of worms recovered from vaccinated animals was significantly shorter than that of the control group. Vaccinated animals showed significantly increased levels of anti-rOv-TSP-2 IgG in the sera after three immunizations, as well as increased levels of several T helper type 1 cytokines in the spleen including IFN-γ and IL-6 but not the Th2/regulatory cytokines IL-4 or IL-10. These results suggest that rOv-TSP-2 could be a potential vaccine against opisthorchiasis and warrants further exploration.

Vaccination of hamsters with Opisthorchis viverrini extracellular vesicles and vesicle-derived recombinant tetraspanins induces antibodies that block vesicle uptake by cholangiocytes and reduce parasite burden after challenge infection.

BACKGROUND: The liver fluke Opisthorchis viverrini infects several million people in Southeast Asia. Adult flukes live in the bile ducts of humans, where they cause hepatobiliary pathology, including cholangiocarcinoma. Here, we investigated the potential of extracellular vesicles (EVs) secreted by the fluke and defined recombinant proteins derived from EVs to generate protective immunity in a hamster vaccination-challenge model. METHODOLOGY/PRINCIPAL FINDINGS: EVs isolated from the excretory-secretory products of O. viverrini and two recombinant EV surface proteins encoding the large extracellular loops (LEL) of Ov-TSP-2 (rOv-TSP-2) and Ov-TSP-3 (rOv-TSP-3) were adjuvanted and used to vaccinate hamsters intraperitoneally followed by challenge infection with O. viverrini metacercariae. The number of adult flukes recovered from hamsters immunized with EVs, rOv-TSP-2, rOv-TSP-3 and rOv-TSP-2+rOv-TSP-3 were significantly reduced compared to control animals vaccinated with adjuvant alone. The number of eggs per gram feces was also significantly reduced in hamsters vaccinated with rOv-TSP-2 compared to controls, but no significant differences were found in the other groups. The average length of worms recovered from hamsters vaccinated with EVs, rOv-TSP-2 and rOv-TSP-3 was significantly shorter than that of worms recovered from the control group. Anti-EV IgG levels in serum and bile were significantly higher in hamsters vaccinated with EVs compared to control hamsters both pre- and post-challenge. In addition, levels of anti-rOv-TSP antibodies in the serum and bile were significantly higher than control hamsters both pre- and post-challenge. Finally, antibodies against rOv-TSP-2 and rOv-TSP-3 blocked uptake of EVs by human primary cholangiocyte in vitro, providing a plausible mechanism by which these vaccines exert partial efficacy and reduce the intensity of O. viverrini infection. CONCLUSION/SIGNIFICANCE: Liver fluke EVs and recombinant tetraspanins derived from the EV surface when administered to hamsters induce antibody responses that block EV uptake by target bile duct cells and exert partial efficacy and against O. viverrini challenge.

In-depth proteomic characterization of Schistosoma haematobium: Towards the development of new tools for elimination.

BACKGROUND: Schistosomiasis is a neglected disease affecting hundreds of millions worldwide. Of the three main species affecting humans, Schistosoma haematobium is the most common, and is the leading cause of urogenital schistosomiasis. S. haematobium infection can cause different urogenital clinical complications, particularly in the bladder, and furthermore, this parasite has been strongly linked with squamous cell carcinoma. A comprehensive analysis of the molecular composition of its different proteomes will contribute to developing new tools against this devastating disease. METHODS AND FINDINGS: By combining a comprehensive protein fractionation approach consisting of OFFGEL electrophoresis with high-throughput mass spectrometry, we have performed the first in-depth characterisation of the different discrete proteomes of S. haematobium that are predicted to interact with human host tissues, including the secreted and tegumental proteomes of adult flukes and secreted and soluble egg proteomes. A total of 662, 239, 210 and 138 proteins were found in the adult tegument, adult secreted, soluble egg and secreted egg proteomes, respectively. In addition, we probed these distinct proteomes with urine to assess urinary antibody responses from naturally infected human subjects with different infection intensities, and identified adult fluke secreted and tegument extracts as being the best predictors of infection. CONCLUSION: We provide a comprehensive dataset of proteins from the adult and egg stages of S. haematobium and highlight their utility as diagnostic markers of infection intensity. Protein composition was markedly different between the different extracts, highlighting the distinct subsets of proteins that different development stages present in their different niches. Furthermore, we have identified adult fluke ES and tegument extracts as best predictors of infection using urine antibodies of naturally infected people. This study provides the first steps towards the development of novel tools to control this important neglected tropical disease.

Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis.

Opisthorchis viverrini Proteome and Host-Parasite Interactions.

The omics technologies have improved our understanding of the molecular events that underpin host-parasite interactions and the pathogenesis of parasitic diseases. In the last decade, proteomics and genomics in particular have been used to characterize the surface and secreted products of the carcinogenic liver fluke Opisthorchis viverrini and revealed important roles for proteins at the host-parasite interface to ensure that the flukes can migrate, feed and reproduce in a hostile environment. This review summarizes the advances made in this area, primarily focusing on discoveries enabled by the publication of the fluke secreted proteomes over the last decade. Protein families that will be covered include proteases, antioxidants, oncogenic proteins and the secretion of exosome-like extracellular vesicles. Roles of these proteins in host-parasite interactions and pathogenesis of fluke-induced hepatobiliary diseases, including cholangiocarcinogenesis, are discussed. Future directions for the application of this knowledge to control infection and disease will also be discussed.

Structural Variants of a Liver Fluke Derived Granulin Peptide Potently Stimulate Wound Healing.

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue ( Ov-GRN12-35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/ trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12-35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12-35_3s and might facilitate the development of a novel wound healing agent.

Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1ß, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.

Polypyridylruthenium(II) complexes exert in vitro and in vivo nematocidal activity and show significant inhibition of parasite acetylcholinesterases.

Over 4.5 billion people are at risk of infection with soil transmitted helminths and there are concerns about the development of resistance to the handful of frontline nematocides in endemic populations. We investigated the anti-nematode efficacy of a series of polypyridylruthenium(II) complexes and showed they were active against L3 and adult stages of Trichuris muris, the rodent homologue of the causative agent of human trichuriasis, T. trichiura. One of the compounds, Rubb12-mono, which was among the most potent in its ability to kill L3 (IC50 = 3.1 ± 0.4 µM) and adult (IC50 = 5.2 ± 0.3 µM) stage worms was assessed for efficacy in a mouse model of trichuriasis by administering 3 consecutive daily oral doses of the drug 3 weeks post infection with the murine whipworm Trichuris muris. Mice treated with Rubb12-mono showed an average 66% reduction (P = 0.015) in faecal egg count over two independent trials. The drugs partially exerted their activity through inhibition of acetylcholinesterases, as worms treated in vitro and in vivo showed significant decreases in the activity of this class of enzymes. Our data show that ruthenium complexes are effective against T. muris, a model gastro-intestinal nematode and soil-transmitted helminth. Further, knowledge of the target of ruthenium drugs can facilitate modification of current compounds to identify analogues which are even more effective and selective against Trichuris and other helminths of human and veterinary importance.

The Venom of the Spine-Bellied Sea Snake (Hydrophis curtus): Proteome, Toxin Diversity and Intraspecific Variation.

The spine-bellied sea snake (Hydrophis curtus) is known to cause human deaths, yet its venom composition has not yet been proteomically characterised. An indepth proteomic analysis was performed on H. curtus venom from two different seasons, January and June, corresponding to adults and subadults, respectively. Venoms from adult and subadult H. curtus individuals were compared using reversedphase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) to detect intraspecific variation, and the molecular weight data obtained with ESIMS were used to assess toxin diversity. RPHPLC and LCESIMS/MS were used to characterise the venom proteome and estimate the relative abundances of protein families present. The most abundant protein family in January and June venoms is phospholipase A2 (PLA2: January 66.7%; June 54.5%), followed by threefinger toxins (3FTx: January 30.4%; June 40.4%) and a minor component of cysteine-rich secretory proteins (CRISP: January 2.5%; June 5%). Trace amounts of snake venom metalloproteinases (SVMP), C-type lectins and housekeeping and regulatory proteins were also found. Although the complexity of the venom is low by number of families present, each family contained a more diverse set of isoforms than previously reported, a finding that may have implications for the development of next-generation sea snake antivenoms. Intraspecific variability was shown to be minor with one obvious exception of a 14,157-Da protein that was present in some January (adult) venoms, but not at all in June (subadult) venoms. There is also a greater abundance of short-chain neurotoxins in June (subadult) venom compared with January (adult) venom. These differences potentially indicate the presence of seasonal, ontogenetic or sexual variation in H. curtus venom.