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

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

Environmental response and adaptation of glycoprotein glue within the droplets of viscous prey capture threads from araneoid spider orb-webs.

Viscous threads that form the prey capture spiral of araneoid orb-webs retain insects that strike the web, giving a spider more time to locate and subdue them. The viscoelastic glycoprotein glue responsible for this adhesion forms the core of regularly spaced aqueous droplets, which are supported by protein axial fibers. Glycoprotein extensibility both facilitates the recruitment of adhesion from multiple droplets and dissipates the energy generated by insects struggling to free themselves from the web. Compounds in the aqueous material make the droplets hygroscopic, causing an increase in both droplet volume and extensibility as humidity (RH) rises. We characterized these humidity-mediated responses at 20%, 37%, 55%, 72% and 90% RH in two large orb-weavers, Argiope aurantia, which is found in exposed habitats, and Neoscona crucifera, which occupies forests and forest edges. The volume-specific extension of A. aurantia glycoprotein reached a maximum value at 55% RH and then declined, whereas that of N. crucifera increased exponentially through the RH range. As RH increased, the relative stress on droplet filaments at maximum extension, as gauged by axial line deflection, decreased in a linear fashion in A. aurantia, but in N. crucifer increased logarithmically, indicating that N. crucifera threads are better equipped to dissipate energy through droplet elongation. The greater hygroscopicity of A. aurantia threads equips them to function in lower RH environments and during the afternoon when RH drops, but their performance is diminished during the high RH of the morning hours. In contrast, the lower hygroscopicity of N. crucifera threads optimizes their performance for intermediate and high RH environments and during the night and morning. These interspecific differences support the hypothesis that viscous capture threads are adapted to the humidity regime of an orb-weaver's habitat.

Humidity affects the extensibility of an orb-weaving spider's viscous thread droplets.

The prey-capture threads found in most spider orb webs rely on viscous droplets for their stickiness. Each droplet is formed of a central mass of viscoelastic glycoprotein glue surrounded by an aqueous covering, both of which incorporate hydrophilic components. We found that the extensibility of droplets on Larinioides cornutus threads increased as humidity increased. However, the deflection of the droplets' supporting axial lines did not change, indicating that atmospheric water uptake increases glycoprotein plasticity, but not glycoprotein adhesion. The extensibility of droplets, along with that of the thread's supporting axial fibers, is responsible for summing the adhesion of multiple thread droplets. Therefore, daily changes in humidity have the potential to significantly alter the performance of viscous threads and orb webs.

Infection with Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Human Cholangiocytes.

Recent reports suggest that the East Asian liver fluke infection, caused by Opisthorchis viverrini, 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, Helicobacter pylori strain NCTC 11637, and the congeneric bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte of the H69 cell line 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. Analysis to quantify cellular proliferation, migration, and invasion in real-time by both H69 cholangiocytes and CC-LP-1 line of cholangiocarcinoma cells using the xCELLigence approach and Matrigel matrix revealed that exposure to 10 H. pylori bacilli per cell stimulated migration and invasion by the cholangiocytes. In addition, 10 bacilli of H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection by H. pylori contributes to the malignant transformation of the biliary epithelium.

Diminished adherence of Biomphalaria glabrata embryonic cell line to sporocysts of Schistosoma mansoni following programmed knockout of the allograft inflammatory factor.

BACKGROUND: Larval 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. METHODS: Bge 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 reverse-transcription-PCR and, in parallel, adhesion of gene-edited Bge cells during co-culture with of schistosome sporocysts was assessed. RESULTS: Gene 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% SD, P ≤ 0.05, n = 12). Adherence by BgAIF gene-edited (ΔBgAIF) 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 ΔBgAIF than control wild type cells adhered to sporocysts; control CAI, 2.66 ± 0.10, ΔBgAIF, 2.30 ± 0.22 (P ≤ 0.01). CONCLUSIONS: The 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.

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.

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.

Differential responses of epithelial cells from urinary and biliary tract to eggs of Schistosoma haematobium and S. mansoni.

Chronic urogenital schistosomiasis can lead to squamous cell carcinoma of the bladder. The International Agency for Research on Cancer classifies the infection with S. haematobium as a group 1 carcinogen, a definitive cause of cancer. By contrast, hepatointestinal schistosomiasis due to the chronic infection with S. mansoni or S. japonicum associated with liver periportal fibrosis, does not apparently lead to malignancy. The effects of culturing human epithelial cells, HCV29, established from normal urothelium, and H69, established from cholangiocytes, in the presence of S. haematobium or S. mansoni eggs were investigated. Cell growth of cells co-cultured with schistosome eggs was monitored in real time, and gene expression analysis of oncogenesis, epithelial to mesenchymal transition and apoptosis pathways was undertaken. Schistosome eggs promoted proliferation of the urothelial cells but inhibited growth of cholangiocytes. In addition, the tumor suppressor P53 pathway was significantly downregulated when exposed to schistosome eggs, and downregulation of estrogen receptor was predicted in urothelial cells exposed only to S. haematobium eggs. Overall, cell proliferative responses were influenced by both the tissue origin of the epithelial cells and the schistosome species.

Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni.

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

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

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

Adhesive compatibility of cribellar and viscous prey capture threads and its implication for the evolution of orb-weaving spiders.

Evolution of orb-weaving spiders that comprise the Orbiculariae clade involved a transition in the composition of prey capture thread that has been challenging to explain. The primitive cribellar threads spun by members of the Deinopoidea subclade resemble the capture threads of their non-orb-web-weaving ancestors and are formed of thousands of fine, dry, protein cribellar fibrils. In contrast, the derived viscous capture threads spun by members of the Araneoidea subclade have regularly spaced, aqueous adhesive droplets. When second instar deinopoid spiderlings emerge from egg sacs they are unable to spin cribellar threads, and, therefore, do not construct orb-webs; whereas second instar araneoids spin capture threads and construct orb-webs. If, as we hypothesize, viscous material evolved to enable second instar spiderlings to construct orb-webs, early araneoids may have spun composite cribellar-viscous capture threads. To examine the functional feasibility of such intermediate capture threads, we compared the adhesion of cribellar threads, viscous threads, and combined cribellar-viscous threads. The stickiness of these combined threads was greater than that of native cribellar or viscous threads alone. The viscous material of Araneus marmoreus threads exhibited a substantial increase in stickiness when combined with cribellar fibrils and that of Argiope aurantia threads a small increase in stickiness when combined with cribellar fibrils. Thus, if early araneoids retained their ability to spin cribellar threads after having evolved glands that produced viscous material, their composite threads could have formed a functional adhesive system that achieved its stickiness at no loss of material economy.