Adult Worm Extracts Research Articles

Moonlighting on the Fasciola hepatica tegument: Enolase, a glycolytic enzyme, interacts with the extracellular matrix and fibrinolytic system of the host.

Enolase is a 47 kDa enzyme that functions within the glycolysis and gluconeogenesis pathways involved in the reversible conversion of D-2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP). However, in the context of host-pathogen interactions, enolase from different species of parasites, fungi and bacteria have been shown to contribute to adhesion processes by binding to proteins of the host extracellular matrix (ECM), such as fibronectin (FN) or laminin (LM). In addition, enolase is a plasminogen (PLG)-binding protein and induces its activation to plasmin, the main protease of the host fibrinolytic system. These secondary 'moonlighting' functions of enolase are suggested to facilitate pathogen migration through host tissues. This study aims to uncover the moonlighting role of enolase from the parasite Fasciola hepatica, shedding light on its relevance to host-parasite interactions in fasciolosis, a global zoonotic disease of increasing concern. A purified recombinant form of F. hepatica enolase (rFhENO), functioning as an active homodimeric glycolytic enzyme of ~94 kDa, was successfully obtained, fulfilling its canonical role. Immunoblotting studies on adult worm extracts showed that the enzyme is present in the tegument and the excretory/secretory products of the parasite, which supports its key role at the host-parasite interface. Confocal immunolocalisation studies of the protein in newly excysted juveniles and adult worms also localised its expression within the parasite tegument. Finally, we showed by ELISA that rFhENO can act as a parasitic adhesin by binding host LM, but not FN. rFhENO also binds PLG and enhances its conversion to plasmin in the presence of the tissue-type and urokinase-type PLG activators (t-PA and u-PA). This moonlighting adhesion-like function of the glycolytic protein enolase could contribute to the mechanisms by which F. hepatica efficiently invades and migrates within its host and encourages further research efforts that are designed to impede this function by vaccination or drug design.

Immunoblotting Identification of Diagnostic Antigens of Paragonimus westermani Type 1 for the Detection of Human Pulmonary Paragonimiasis in North East India.

Human pulmonary paragonimiasis, an emerging concern in North East India, frequently masquerades as pulmonary tuberculosis due to clinical and radiological similarities, leading to diagnostic challenges. This research aimed to harness the immunoblotting technique to discern immunodiagnostic protein antigens from both adult worm and excretory-secretory (ES) extracts of the prevalent Paragonimus westermani type 1 in Arunachal Pradesh, North East India. We studied the time kinetics of immunoreactive patterns in relation to the duration of infection in rodent models. Immunoblot analyses were also conducted using sera from ELISA-positive patients confirmed with paragonimiasis, facilitating the selection of antigenic extracts with diagnostic potential. Further, ES protein antigens were subjected to 2D immunoblot analysis and immunoreactive protein spots identified using MALDI-TOF MS. The immunoreactivity patterns of ES antigens with sera of paragonimiasis-positive patients were detailed, and specific immunoreactive protein antigens were pinpointed using peptide mass fingerprinting (MALDI-TOF). This work underscores the enhanced diagnostic accuracy when combining ELISA with immunoblotting for pulmonary paragonimiasis in regions like North East India, marked by co-existing helminth infections.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator.

Screening of Capillaria philippinensis infection using Trichuris muris and Trichinella spiralis antigens

Background: Intestinal capillariasis is a disease caused by Capillaria philippinensis. Human infectionsbecame more prevalent in many countries including Egypt. This nematode is related to Trichinella andTrichuris species, all of them belong to Trichinelloidea superfamily. Diagnosis of intestinal capillariasismay be missed by stool examination.Objective: This study aimed to use enzyme-linked immunosorbent assay (ELISA) and immunoblotting inscreening for intestinal capillariasis as a practical and rapid diagnostic test.Subjects and Methods: Trichuris muris and Trichinella spiralis adult worms were isolated from infectedmice and crude antigens were prepared. The protein content for both adult worm extracts was determined.Human blood samples were collected from 20 capillariasis patients, 20 control individuals and 10 W.bancrofti infected patients. The study evaluated cross-reactivity between T. spiralis and T. muris antigensand sera from cases infected with C. philippinensis using ELISA and immunoblotting.Results: In ELISA, T. muris crude antigen cross reacted with 100% of capillariasis sera with 100%sensitivity and specificity and cross reacted with 10% of sera from bancroftian filariasis. T. spiralis crudeantigen cross reacted with 50% of capillariasis sera, and 9% of sera from bancroftian filariasis. Neither T.muris nor T. spiralis crude antigens reacted with sera from control group. Immunoblotting results showedthat IgG antibodies from control group didn't recognize specific proteins in T. muris antigen, while in W.bancrofti group, only one band in one sample appeared (100-135 kDa). IgG antibodies from capillariasiscases recognized multiple common protein bands (35-180 kDa). IgG antibodies from capillariasis, controland W. bancrofti sera did not recognize specific proteins in T. spiralis crude antigen.Conclusion: Antigens from T. muris and T. spiralis can be used successfully to detect infection with C.philippinensis using serum samples of cases by ELISA. Crude antigen of T. muris gave better results thanthat of T. spiralis. Immunoblotting can be used for diagnosis of capillariasis by using crude antigen of T.muris.

A novel in vitro Caenorhabditis elegans transcription system

BackgroundCaenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system.ResultsBy employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner.ConclusionsIn this study, we developed an in vitro C. elegans transcription system that re-constitutes transcription reactions with nuclear extract of larval or adult worms and can both qualitatively and quantitatively detect transcription activity using non-radioactive approaches. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

Deep phosphoproteome analysis of Schistosoma mansoni leads development of a kinomic array that highlights sex-biased differences in adult worm protein phosphorylation.

Although helminth parasites cause enormous suffering worldwide we know little of how protein phosphorylation, one of the most important post-translational modifications used for molecular signalling, regulates their homeostasis and function. This is particularly the case for schistosomes. Herein, we report a deep phosphoproteome exploration of adult Schistosoma mansoni, providing one of the richest phosphoprotein resources for any parasite so far, and employ the data to build the first parasite-specific kinomic array. Complementary phosphopeptide enrichment strategies were used to detect 15,844 unique phosphopeptides mapping to 3,176 proteins. The phosphoproteins were predicted to be involved in a wide range of biological processes and phosphoprotein interactome analysis revealed 55 highly interconnected clusters including those enriched with ribosome, proteasome, phagosome, spliceosome, glycolysis, and signalling proteins. 93 distinct phosphorylation motifs were identified, with 67 providing a ‘footprint’ of protein kinase activity; CaMKII, PKA and CK1/2 were highly represented supporting their central importance to schistosome function. Within the kinome, 808 phosphorylation sites were matched to 136 protein kinases, and 68 sites within 37 activation loops were discovered. Analysis of putative protein kinase-phosphoprotein interactions revealed canonical networks but also novel interactions between signalling partners. Kinomic array analysis of male and female adult worm extracts revealed high phosphorylation of transformation:transcription domain associated protein by both sexes, and CDK and AMPK peptides by females. Moreover, eight peptides including protein phosphatase 2C gamma, Akt, Rho2 GTPase, SmTK4, and the insulin receptor were more highly phosphorylated by female extracts, highlighting their possible importance to female worm function. We envision that these findings, tools and methodology will help drive new research into the functional biology of schistosomes and other helminth parasites, and support efforts to develop new therapeutics for their control.

Extract of Ascaris suum induces TGF-β and early production of IgG1 in experimental autoimmune hepatitis.

In experimental autoimmune hepatitis (EAH) of Th1 profile, an extract of adult Ascaris suum worms (ASC) was previously found to deviate the immune response to a Th2/IL-10 pattern. Here, the effects of treatment with ASC on production of TGF-β and the anti-Ascaris isotypes IgG1 and IgG2a in EAH were evaluated. EAH was induced in BALB/c mice, intravenously with concanavalin A. Two hours later, these animals received ASC (EAH+ASC group) or PBS vehicle (EAH group). IgG1 and IgG2a were evaluated 8 h, 24 h and 7 d after induction. TGF-β was measured in a splenocyte culture at this last time. The isotype levels in the EAH group were low throughout the kinetics. In the EAH+ASC group, there was significant production of IgG1 at 24 h and 7 d, but of IgG2a only at 7 d. There was statistically greater production of TGF-β in the EAH+ASC group. The higher levels of IgG1 and TGF-β in this group suggest that an additional Th1 response control route exists in EAH, which needs to be investigated.

Adiponectin Limits IFN-γ and IL-17 Producing CD4 T Cells in Obesity by Restraining Cell Intrinsic Glycolysis.

Compared to the innate immune system, the contribution of the adaptive immune response during obesity and insulin resistance is still not completely understood. Here we demonstrate that high fat diet (HFD) increases the frequencies of activated CD4+ and CD8+ T cells and frequencies of T cells positive for IFN-γ and IL-17 in the adipose tissue. The adipocyte-derived soluble factor adiponectin reduces IFN-γ and IL-17 positive CD4+ T cells from HFD mice and dampens the differentiation of naïve T cells into Th1 cells and Th17 cells. Adiponectin reduces Th17 cell differentiation and restrains glycolysis in an AMPK dependent fashion. Treatment with adult worm extracts of the rodent filarial nematode Litomosoides sigmodontis (LsAg) reduces adipose tissue Th1 and Th17 cell frequencies during HFD and increases adiponectin levels. Stimulation of T cells in the presence of adipocyte-conditioned media (ACM) from LsAg-treated mice reduces Th1 and Th17 frequencies and this effect was abolished when ACM was treated with an adiponectin neutralizing antibody. Collectively, these data reveal a novel role of adiponectin in controlling pro-inflammatory CD4+ T cells during obesity and suggest that the beneficial role of helminth infections and helminth-derived products on obesity and insulin resistance may be in part mediated by adiponectin.

Preventive and therapeutic effects of Trichinella spiralis adult extracts on allergic inflammation in an experimental asthma mouse model

BackgroundHelminths immunomodulate the host immune system by secreting proteins to create an inhibitory environment as a strategy for survival in the host. As a bystander effect, this balances the host immune system to reduce hypersensitivity to allergens or autoantigens. Based on this, helminth therapy has been used to treat some allergic or autoimmune diseases. As a tissue-dwelling helminth, Trichinella spiralis infection has been identified to have strong immunomodulatory effects; the effective components in the worm have not yet been identified.MethodsThe soluble extracts of T. spiralis adult worms and muscle larvae were used to treat airway inflammation before and after an ovalbumin (OVA)-sensitization/challenge in an OVA-induced asthma mouse model. The therapeutic effects were observed by measuring the level of inflammation in the lungs.ResultsThe soluble products derived from T. spiralis parasites, especially from adult worms, were able to ameliorate OVA-induced airway inflammatory responses which were associated with reduced eosinophil infiltration, OVA-specific IgE, Th2 cytokine IL-4, and increased IL-10 and TGF-β. The stimulation of the Treg response may contribute to the alleviated allergic inflammation.ConclusionsTrichinella spiralis worm extracts stimulate regulatory cytokines that are associated with reduced allergic airway inflammation. The identification of effective components in the adult worm extracts will be a crucial approach for developing a novel therapeutic for allergic and autoimmune diseases.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms

BackgroundAngiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells.MethodsThe expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls.ResultsFirst, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections.ConclusionsThe ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.

Identification and molecular characterization of exosome-like vesicles derived from the Taenia asiatica adult worm

Taenia asiatica is an important food-borne parasite that poses a threat to food-safety and animal husbandry hygine, yet little is known about its specific infection and immune escape mechanisms. Exosome-like vesicles have recently emerged as a regulator in the interactions between parasites and hosts, providing a new direction for research on infection of T. asiatica. In this experiment, exosome-like vesicles were collected from the excretory/secretory products of cultured T. asiatica and isolated by differential centrifugation. The purified vesicles, ranging from 30 to 150 nm in size, were identified as exosome-like vesicles by transmission electron microscope and Nanoparticle tracking analysis. Proteomics analysis identified 455 proteins in the exosome-like vesicles. Of these proteins, enzymes involved in metabolic processes were identified, including glyceraldehyde 3 phosphate dehydrogenase, fructose-1, 6-bisphosphate aldolase, cytosolic malate dehydrogenase, and enolase. The two most abundant proteins from proteomic analysis, 14-3-3 and enolase, were shown to be present in the exosome-like vesicles by immunogold labeling. High-throughput RNA sequencing yielded twenty known miRNAs present in exosome-like vesicle sRNA libraries. Nine of the miRNAs, including six known miRNAs (tas-miR-71, tas-miR-1, tas-miR-7, tas-miR-9, tas-miR-10, and tas-let-7) and three newly discovered miRNAs (tas-m0022-3p, tas-m0816-3p, tas-m0082-5p), were confirmed by RT-qPCR as present in T. asiatica adult worm extracts and secreted exosome-like vesicles in T. asiatica. Additionally, we demonstrated that exosome-like vesicles experimentally labeled with PKH67 were internalized by LoVo cells in vitro. These findings provide new insights into the interaction between tapeworms and hosts mediated by exosome-like vesicles.

Heat shock protein 70 of filarial parasite Setaria equina: Cloning, expression, and analysis of binding with diethylcarbamazine citrate

Setaria equina heat shock protein (SeqHSP) 70 gene was characterized, cloned and expressed to recombinant protein (rSeqHSP70). The protein was tested for binding with an anti-filarial drug “diethylcarbamazine citrate (DEC)” by equilibrium dialysis method. Molecular docking was also used to determine the binding sites and residues of binding with DEC. The mice were immunized with the protein alone or bound to DEC. Serum IFN-γ levels in the immunized group with protein-drug complex were significantly higher (P < 0.05) than the protein-immunized group. Mouse anti-SeqHSP70 polyclonal IgG recognized 2 bands at 70 and 75 kDa in S. equina adult worm and human cancer cell lines (HepG2 and MCF-7) extracts. The proliferation assay for mice splenocytes revealed a potentiation and down-regulating effects in non-immunized and immunized groups, respectively with the drug-protein complex. The proliferation and IFN-γ assays for purified human NK cells indicated a potentiating effect of the drug-protein complex (DEC concentration is 50 μM) comparable to the protein. DEC at lower concentration (25 mM) could also show a significant increase (P < 0.05) in IFN-γ. From the results, DEC was postulated to induce conformational changes in the protein exposing more epitopes for NK cell binding and activation.

Filarial extract of Litomosoides sigmodontis induces a type 2 immune response and attenuates plaque development in hyperlipidemic ApoE-knockout mice.

A type 1 immune response is involved in atherosclerosis progression, whereas the role of a type 2 polarization, especially with regard to an enhanced T helper (Th)2 cell differentiation, is still unclear. Helminths trigger type 2 immune responses, protecting the host from inflammatory disorders. We investigated whether an increased type 2 polarization by administration of Litomosoides sigmodontis adult worm extract (LsAg) affects atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. Injections of 50 µg LsAg, i.p. into ApoE-/- mice induced a type 2 immune response shown by increased frequencies of peritoneal eosinophils and alternatively activated macrophages. To analyze the effect of LsAg on atherosclerosis initiation, ApoE-/- mice received a high-fat diet for 12 wk and weekly injections of 50 µg LsAg from wk 5 to 12. Therapeutic effects on advanced atherosclerosis were analyzed in mice that were fed a high-fat diet for 12 wk followed by 12 wk of normal chow and weekly LsAg injections. Both preventive and therapeutic LsAg application significantly decreased plaque size. Therapeutic treatment even caused regression of plaque size and macrophage density in the aortic root and reduced Th1-specific gene expression and intraplaque inflammation. In addition, plaque size after therapeutic treatment was inversely correlated with plaque-infiltrated alternatively activated macrophages. In vitro, LsAg treatment of HUVECs reduced intracellular levels of phosphorylated NF-κB-p65, IκB-α, and JNK1/2. In bifurcation flow-through slides, THP-1 cell adhesion to a HUVEC monolayer was decreased by LsAg in regions of nonuniform shear stress. Applying inhibitors of the respective kinases suggests JNK1/2 inhibition is involved in the suppressed cell adhesion. A switch to an enhanced type 2 immune response by LsAg exerts antiatherogenic effects on murine plaque development, indicating a protective role of a hampered type 1 polarization. In vitro, LsAg affects endothelial signaling pathways, among which JNK1/2 inhibition seems to be involved in the suppression of monocytic cell adhesion under proatherogenic shear stress.-Constanze, K., Tauchi, M., Furtmair, R., Urschel, K., Raaz-Schrauder, D., Neumann, A.-L., Frohberger, S. J., Hoerauf, A., Regus, S., Lang, W., Sagban, T. A., Stumpfe, F. M., Achenbach, S., Hübner, M. P., Dietel, B. Filarial extract of Litomosoides sigmodontis induces a type 2 immune response and attenuates plaque development in hyperlipidemic ApoE-knockout mice.

Treatment of Schistosoma mansoni with miltefosine in vitro enhances serological recognition of defined worm surface antigens.

BackgroundMiltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro.Methodology/Principal findingsIn an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH). Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS) combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA), Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection.Conclusion/SignificanceThis study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ), whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug’s anti-parasite efficacy and be involved in the development of resistance to re-infection.

Identification and functional characterisation of a Schistosoma japonicum insulin-like peptide

BackgroundPrevious studies have shown that insulin receptors in schistosomes, triggered by host insulin, play an important role in parasite growth, development and fecundity by regulating glucose metabolism. However, limited information is available on the recently identified endogenous insulin-like peptide (ILP) in blood flukes.ResultsWe isolated ILPs from Schistosoma japonicum (SjILP) and S. recognised (SmILP) and present results of their molecular and structural analysis. SjILP shares 63% amino acid identity with SmILP, but only 18% identity with human insulin. There is high cross immunological reactivity between the S. japonicum and S. mansoni ILPs as observed in western blots using an anti-SjILP polyclonal antibody. ADP binding/hydrolysis ability was observed in both SjILP and SmILP, but not in human insulin, suggesting a parasite-specific role for ILP compared with host insulin. Protein binding assays using the Octet-RED system showed SjILP binds S. japonicum IRs (SjIR1 and SjIR2) strongly. An anti-phospho antibody against extracellular signal-regulated kinase (Erk) recognised a 44-kDa target band in an extract of adult worms after stimulation by rSjILP in vitro, suggesting an important role for SjILP in activating SjIRs and in regulating downstream signal transduction. Immunolocalisation showed SjILP is located on the tegument and the underlying musculature, similar to that observed for SjIR1, but it is also present throughout the parenchyma of males and in the vitelline cells of females, the same locations as SjIR2 described in an earlier published study of ours. The same localisation of SjILP and the SjIRs is suggestive of an interaction between the insulin-like peptide and the IRs. In addition to binding host insulin, schistosomes also can express their own endogenous ILPs, which can activate the parasite insulin signal pathway, thereby playing a critical role in worm growth, development and fertility.ConclusionsThese findings shed new light on ILPs in schistosomes, providing further insight into the distinct and specialised functions of SjIR1 and 2 in S. japonicum and their interaction with host insulin.

Comparing antibody responses to &lt;i&gt;Onchocerca volvulus&lt;/i&gt; and non-parasite antigens in placebo-controlled and ivermectin-treated onchocerciasis patients

Serum antibodies to parasite-specific and non-parasite antigens were evaluated using enzyme-linked immunosorbent assay (ELISA). Out of the 470 sera collected, 409 were from residents of an onchocerciasis hyper-endemic area, 55 non-endemic and 6 European normal sera served as control. The patients’ age, sex, skin microfilaria densities, dermal and ocular clinical manifestations (colour of optic disc) have been well characterised. The study population had participated in a placebocontrolled (n=191) trial of ivermectin (Mectizan®) treatment (n=218). The parasite antigens are phosphate buffered saline crude extract of adult worms of Onchocerca volvulus, a recombinant antigen (Ov1.9) and a monoclonal antibody purified antigen (Cam 1). The non-parasite antigens are deoxycholate citrate extract of optic nerve (nerve-DOC) and commercially available IgA, IgM and IgG were used to assay for rheumatoid factor (Rh-F) auto-antibodies. Generally, antibodies to parasite antigens in onchocerciasis patients were remarkably higher than control group (p&lt;0.05) using exact F-test. There was no significant difference (p&gt;0.05) in antibodies to nerve-DOC and Rh-F in patients compared to control. Antibodies increased with increasing skin snip microfilaria load from 0.69±0.28 with 0mf/mg (n=54) as against 0.80±0.26 for those with 4-20mf/mg. Observed slight negative correlation in IgG antibody levels and severity of disc colour with mean OD values of 0.26±0.22 in those graded as having no optic nerve disease (OND) (disc 1, n=86) and 0.17±0.19 for those with severe changes (disc 3, n=49) was not statistically significant (P&gt;0.05). An age dependent significant decrease (P&lt;0.05) in antibodies were observed with 0.64±0.34 for 15-30yr old (n=48) compared to 0.48±0.35 for those 50yr (n=50) for PBS with a similar trend for IgG to Ov1.9 and Cam1. In conclusion, serum parasite-specific and non-parasite antibodies may not be responsible for the pathology of optic nerve disease. Onchocerciasis patients were apparently not at higher risk of developing rheumatoid arthritis than the control.Keywords: Onchocerciasis; Antibodies; Antigens; Immune responses; Ivermectin.

High-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) reveals Argonaute-associated microRNAs and targets in Schistosoma japonicum.

BackgroundSchistosomiasis, caused by schistosomes, is one of the most prevalent and serious parasitic diseases in tropical and subtropical countries. This pathogen has a complex life cycle and harbors a unique repertoire of genes expressed at different life-stages. Understanding the gene regulation of schistosomes will contribute to identification of novel drug targets and vaccine candidates. Some conserved and novel microRNAs (miRNAs) have been identified in schistosomes as key transcriptional and post-transcriptional regulators in the past few years; however, little is known about their specific targets.MethodsHigh-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation (HITS-CLIP) was used to covalently crosslink native Argonaute protein-RNA complexes in Schistosoma japonicum. An antibody against S.japonicum Argonaute proteins, was generated and used for immunoprecipitation of the crosslinked SjAgo-RNA complex from soluble adult worm extract. Small RNAs, including miRNAs and their target mRNAs associated with the native SjAgo in adult parasites, were enriched and extracted for library construction.ResultsHigh-throughput sequencing produced a total of ~7.4 million high-quality reads, of which approximately 45.07 % were composed of 769 miRNAs and 35.54 % were composed of 11,854 mRNAs target sites. Further bioinformatics analysis identified 43 conserved known miRNAs and 256 novel miRNAs in the SjAgo-associated small RNA population. An average of approximately 15 target sites were predicted for each miRNA. Moreover, a positive rate of 50 % has been achieved in a small-scale verification test of the putative target sites of miRNA1.ConclusionIn this study, we isolated and identified small RNAs including miRNAs and their targets associated with the S. japonicum Argonaute proteins, by the HITS-CLIP method combined with bioinformatics and biologic experimental analysis. These data reveal a genome-wide miRNA-mRNA interaction map in S. japonicum in vivo, which will help us understand the complex gene regulatory network in this pathogen and thereby facilitate the development of novel drug approaches against schistosomiasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1203-9) contains supplementary material, which is available to authorized users.

Molecular cloning and characterization of a nematode polyprotein antigen/allergen from the human and animal hookworm Ancylostoma ceylanicum

Nematodes are unable to synthesize fatty acids de novo and must acquire them from the environment or host. It is hypothesized that two unique classes of fatty acid and retinol binding proteins that nematodes produce (fatty acid and retinol binding (FAR) and nematode polyprotein antigen/allergen (NPA)) are used to meet this need. A partial cDNA has been cloned corresponding to four subunits of a putative Ancylostoma ceylanicum NPA (AceNPA). The translated amino acid sequence of AceNPA shares sequence identity with similar proteins from Dictyocaulus viviparus, Ascaris suum, and Ostertagia ostertagi. Immunoblot experiments using a polyclonal anti-AceNPA IgG revealed proteins corresponding to the expected sizes of single, as well as two or three un-cleaved NPA subunits in adult excretory/secretory proteins and soluble adult worm extracts. Immunohistochemistry experiments localize AceNPA to the cuticle, pseudocoelomic space and testes suggesting a role in hookworm biology that is distinct from what has previously been defined for other hookworm lipid binding proteins. A single recombinant subunit of AceNPA (rAceNPAb) demonstrated binding in vitro to fluorescent fatty acids DAUDA, cis-parinaric acid, as well as retinol, at equilibrium dissociation constants in the low micromolar range. Further, in vitro data reveal that rAceNPAb binds fatty acids with chain lengths of C12–C22, with the greatest affinities for arachidonic, linoleic (C18), and eicosapentaenoic (C20) acids.

Wolbachia lipoproteins: abundance, localisation and serology of Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6 from Brugia malayi and Aedes albopictus.

BackgroundLipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery. Here we characterise the abundance, localisation and serology of the Wolbachia lipoproteins: Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6.MethodsWe used proteomics to confirm lipoprotein presence and relative abundance; fractionation, immunoblotting and confocal and electron immuno-microscopy for localisation and ELISA for serological analysis.ResultsProteomic analysis of Brugia malayi adult female protein extracts confirmed the presence of two lipoproteins, previously predicted through bioinformatics: Wolbachia peptidoglycan associated lipoprotein (wBmPAL) and the Type IV Secretion System component, VirB6 (wBmVirB6). wBmPAL was among the most abundant Wolbachia proteins present in an extract of adult female worms with wBmVirB6 only detected at a much lower abundance. This differential abundance was reflected in the immunogold-labelling, which showed wBmPAL localised at numerous sites within the bacterial membranes, whereas wBmVirB6 was present as a single cluster on each bacterial cell and also located within the bacterial membranes. Immunoblotting of fractionated extracts confirmed the localisation of wBmPAL to membranes and its absence from cytosolic fractions of C6/36 mosquito cells infected with wAlbB. In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia. Serological analysis showed that both proteins were immunogenic and raised antibody responses in the majority of individuals infected with Wuchereria bancrofti.ConclusionsTwo Wolbachia lipoproteins, wBmPAL and wBmVirB6, are present in extracts of Brugia malayi with wBmPAL among the most abundant of Wolbachia proteins. Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.

Two SmDLC antigens as potential vaccines against schistosomiasis

The Schistosoma mansoni transcriptome revealed new members of the dynein light chain family (DLC/LC8). The antigenicity and immunogenicity of these proteins, and their potential as vaccine candidates were investigated. Two DLC genes (DLC12_JI392413.1 and DLC13_JI387686.1) were cloned and the recombinant proteins produced in E. coli. The immunization of mice with the rDLCs, using alhydrogel as adjuvant, resulted in high titers of antibodies, indicated that these proteins are highly immunogenic. The anti-DLCs antibodies presented cross reactivity with both recombinant antigens and also recognized proteins from S. mansoni adult worm extracts. The DLC12 and DLC13 immunized animals were challenged by infection with cercariae and a protective profile was observed in three different assays, with a significant decreased in worm burden, of 43% and 51% respectively, when compared to the non-vaccinated group. The granulomas formation due to egg retention in the hepatic tissues was evaluated 45 days after infection. Smaller granulomas were observed in the liver of DLC immunized animals, up to 70% reduction in comparison to the granulomas size in the non-vaccinated animals. Fifty-five days after infection, the average size of the hepatic granulomas was still 25–35% smaller in the DLCs vaccinated groups. The interference of DLC immunization on the hepatic granuloma formation may reflect the lower worm burden and consequent decrease on the number of eggs retained in the liver, resulting in lower pro-inflammatory level in the tissue.The protective effect of DLCs immunization, decreasing the worm burden and delaying the rate of granuloma formation, suggests that these antigens should be further studied as potential vaccine candidates.