Excretory-secretory Products Research Articles

METABOLIC ADAPTATION OF FISH INTESTINAL HELMINTHS: INHIBITORY ABILITY TOWARDS PROTEASES IN CESTODES <i>TRIAENOPHORUS NODULOSUS</i>

The inhibitory ability towards proteases was studied in the extracts of plerocercoids from the liver of perch (Perca fluviatilis Linnaeus), as well as in the excretory-secretory products and extracts of mature and immature cestodes Triaenophorus nodulosus (Pallas, 1781) from the intestine of pike (Esox lucius Linnaeus). It was found that excretory-secretory products and extracts of both mature and immature T. nodulosus display varying degrees of inhibitory ability against proteases. A significant decrease in activity was noted under the action of the studied samples on the activity of commercial trypsin. Tapeworm extracts exhibit a greater inhibitory ability compared to excretory-secretory products. At the same time, the extract of immature worms inhibits the activity of proteases more strongly than that of mature ones. The inhibitory ability is more closely associated with the extract of worms lacking the brush border than with the tegumental brush border fraction.

The proteome of extracellular vesicles of the lung fluke Paragonimus kellicotti produced in vitro and in the lung cyst

Paragonimiasis is a zoonotic, food-borne trematode infection that affects 21 million people globally. Trematodes interact with their hosts via extracellular vesicles (EV) that carry protein and RNA cargo. We analyzed EV in excretory-secretory products (ESP) released by Paragonimus kellicotti adult worms cultured in vitro (EV ESP) and EV isolated from lung cyst fluid (EV CFP) recovered from infected gerbils. The majority of EV were approximately 30–50 nm in diameter. We identified 548 P. kellicotti-derived proteins in EV ESP by mass spectrometry and 8 proteins in EV CFP of which 7 were also present in EV ESP. No parasite-derived proteins were reliably detected in EV isolated from plasma samples. A cysteine protease (MK050848, CP-6) was the most abundant protein found in EV CFP in all technical and biological replicates. Immunolocalization of CP-6 showed strong labeling in the tegument of P. kellicotti and in the adjacent cyst and lung tissue that contained worm eggs. It is likely that CP-6 present in EV is involved in parasite-host interactions. These results provide new insights into interactions between Paragonimus and their mammalian hosts, and they provide potential clues for development of novel diagnostic tools and treatments.

Regulation of immune response against third-stage Gnathostoma spinigerum larvae by human genes.

Gnathostomiasis is an important zoonosis in tropical areas that is mainly caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3). This study aimed to prove whether G. spinigerum L3 produces extracellular vesicles (EVs) and investigate human gene profiles related to the immune response against the larvae. We created an immune cell model using normal human peripheral blood mononuclear cells (PBMCs) co-cultured with the larvae for 1 and 3 days, respectively. The PBMCs were harvested for transcriptome sequencing analysis. The EV ultrastructure was examined in the larvae and the cultured medium. Extracellular vesicle-like particles were observed under the larval teguments and in the pellets in the medium. RNA-seq analysis revealed that 2,847 and 3,118 genes were significantly expressed on days 1 and 3 after culture, respectively. The downregulated genes on day 1 after culture were involved in pro-inflammatory cytokines, the complement system and apoptosis, whereas those on day 3 were involved in T cell-dependent B cell activation and wound healing. Significantly upregulated genes related to cell proliferation, activation and development, as well as cytotoxicity, were observed on day 1, and genes regulating T cell maturation, granulocyte function, nuclear factor-κB and toll-like receptor pathways were predominantly observed on day 3 after culture. G. spinigerum L3 produces EV-like particles and releases them into the excretory-secretory products. Overall, genotypic findings during our 3-day observation revealed that most significant gene expressions were related to T and B cell signalling, driving T helper 2 cells related to chronic infection, immune evasion of the larvae, and the pathogenesis of gnathostomiasis. Further in-depth studies are necessary to clarify gene functions in the pathogenesis and immune evasion mechanisms of the infective larvae.

The therapeutic effect of tanshinone IIA in mouse astrocytes after treatment with Angiostrongylus cantonensis fifth-stage larval excretory-secretory products.

Angiostrongylus cantonensis is an important food-borne zoonotic parasite that causes eosinophilic meningitis and meningoencephalitis in humans. Excretory-secretory products (ESPs) are valuable targets for studying host-parasite relationships. ESPs are composed of a variety of molecules that are used to penetrate defensive barriers and avoid immune attack of the host. Tanshinone IIA (TSIIA) is a vasoactive cardioprotective drug that is widely used in studies evaluating potential therapeutic mechanisms. In this study, we will evaluate the therapeutic effects of TSIIA in mouse astrocytes after A. cantonensis fifth-stage larvae (L5) ESPs treatment. Here, we examined the therapeutic effect of TSIIA by real-time qPCR, western blotting, activity assay, and cell viability assays. First, the results showed that TSIIA can elevate cell viability in astrocytes after stimulation with ESPs. On the other hand, TSIIA downregulated the expression of apoptosis-related molecules. However, the expression of molecules related to antioxidant, autophagy, and endoplasmic reticulum stress was significantly increased. The results of antioxidant activation assays showed that the activities of superoxide dismutase (SOD), glutathione S-transferase (GST), and catalase were significantly increased. Finally, we found that cell apoptosis and oxidative stress were reduced in TSIIA-treated astrocytes by immunofluorescence staining. The findings from this study suggest that TSIIA can reduce cellular damage caused by A. cantonensis L5 ESPs in astrocytes and clarify the related molecular mechanisms.

Recombinant protein EBI3 attenuates Clonorchis sinensis-induced liver fibrosis by inhibiting hepatic stellate cell activation in mice

BackgroundChronic infection with Clonorchis sinensis can cause hepatobiliary fibrosis and even lead to hepatobiliary carcinoma. Epstein-Barr virus-induced gene 3 protein (EBI3) is a subunit of interleukin 35, which can regulate inflammatory response and the occurrence of fibrotic diseases. Previous studies have reported that the expression of EBI3 in the serum of patients with liver cirrhosis is reduced. The present study aims to investigate the biological effects of EBI3 on liver fibrosis caused by C. sinensis and the underlying molecular mechanisms.MethodsWe first established a mouse model of liver fibrosis induced by C. sinensis infection and then measured the serum expression of EBI3 during the inflammatory and fibrotic phase. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to investigate the potential role of EBI3 in liver fibrosis by regulating the extracellular matrix structural constituent and collagen catabolic process. Recombinant protein EBI3 (rEBI3) was added to hepatic stellate cells (HSCs) in vitro with C. sinensis antigen to explore its function. Finally, the therapeutic effect of rEBI3 was verified by intravenous injection into C. sinensis-infected mice.ResultsThe results showed that the serum expression of EBI3 increased in the inflammatory response phase but decreased in the fibrotic phase. The excretory-secretory products of C. sinensis (Cs.ESP) were able to stimulate HSC activation, while rEBI3 reduced the activation of HSCs induced by Cs.ESP. Also, the protein expression of gp130 and downstream protein expressions of JAK1, p-JAK1, STAT3 and p-STAT3 in HSCs were increased after rEBI3 incubation. Finally, intravenously injected rEBI3 inhibited hepatic epithelial-mesenchymal transition in C. sinensis-infected mice by inhibiting HSC activation and reducing liver injury.ConclusionThis study confirms that rEBI3 can attenuate C. sinensis-induced liver fibrosis by inhibiting HSC activation and may be one of the potential treatments for liver fibrosis.Graphical

Cystatins from the Human Liver Fluke Opisthorchis viverrini: Molecular Characterization and Functional Analysis.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

The regulatory roles of Fasciola hepatica GSTO1 protein in inflammatory cytokine expression and apoptosis in murine macrophages

Fascioliasis, a global zoonotic parasitic disease, is mainly caused by Fasciola hepatica (F. hepatica) parasitizing in the livers of hosts, mainly humans and herbivores. Glutathione S-transferase (GST) is one of the important excretory- secretory products (ESPs) from F. hepatica, however, the regulatory roles of its Omega subtype in the immunomodulatory effects remain unknown. Here, we expressed F. hepatica recombinant GSTO1 protein (rGSTO1) in Pichia pastoris and analyzed its antioxidant properties. Then, the interaction between F. hepatica rGSTO1 and RAW264.7 macrophages and its effects on inflammatory responses and cell apoptosis were further explored. The results revealed that GSTO1 of F. hepatica owned the potent ability to resist oxidative stress. F. hepatica rGSTO1 could interact with RAW264.7 macrophages and inhibit its cell viability, furthermore, it may suppress the production of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, but promote the expression of anti-inflammatory cytokine IL-10. In addition, F. hepatica rGSTO1 may down-regulate the ratio of Bcl-2/Bax, and increase the expression of pro-apoptotic protein caspase-3, thereby eliciting the apoptosis of macrophages. Notably, F. hepatica rGSTO1 inhibited the activation of nuclear factor-κB (NF-κB) and mitogen‑activated protein kinases (MAPKs p38, ERK and JNK) pathways in LPS-activated RAW264.7 cells, exerting potent modulatory effects on macrophages. These findings suggested that F. hepatica GSTO1 can modulate the host immune response, which provided new insights into the immune evasion mechanism of F. hepatica infection in host.

Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.

Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite Brugia malayi to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.

In vitro knockdown of TsDNase II-7 suppresses Trichinella spiralis invasion into the host's intestinal epithelial cells.

Trichinella spiralis (T. spiralis) adult-specific deoxyribonuclease II-7 (TsDNase II-7), a member of the DNase II-like nuclease family with no DNase II activity, was identified in the excretory-secretory (ES) products of adult worms (AWs). However, its biological functions are still unclear. Our previous study revealed that TsDNase II-7 is located around the infection site in the intestinal tissue, speculating that it was involved in the T. spiralis invasion of host intestinal epithelial cells (IECs). This study aimed to use RNA interference to verify our speculation that TsDNase II-7 in 3-day old adult T. spiralis (Ad3) plays a role in intestinal invasion. TsDNase II-7-specific small interfering RNAs (siRNAs) were delivered into muscle larvae (MLs) to knockdown TsDNase II-7 expression by electroporation. Twenty-four hours later, the MLs transfected with 2 μM siRNA-841 exhibited decreased in TsDNase II-7 transcription and expression as compared to the control MLs. The knockdown of TsDNase II-7 expression did not affect ML viability, and the low expression of TsDNase II-7 still maintained in Ad3 recovered from TsDNase II-7-RNAi-ML infected mice, resulting in a weakened ability of Ad3 to invade intestinal epithelial cells (IECs). These results indicated that knockdown of TsDNase II-7 gene expression via RNA interference (RNAi) suppressed adult worm invasion and confirmed that TsDNase II-7 plays a crucial role during the intestinal phase of T. spiralis infections, which provided new candidate for vaccine development of T. spiralis.

Fasciola hepaticaFh15 promote survival in a mouse septic shock model and downregulates inflammatory cytokines

Abstract Parasitic helminths and helminth-derived molecules, possess an inherent anti-inflammatory capacity. Fasciola hepatica has been reported to decrease in vivo particular Th1 immune responses caused by concurrent bacterial infections. Studies have shown that some Fasciola hepatica’s excretory-secretory products has the capacity to reduce phagocytic activity, affect cell recruitment, and induce activation of alternative activated macrophages. We hypothesized that recombinant F. hepatica FABP (Fh15) may be an excellent target for the development of new anti-inflammatory drugs against sepsis. For this, we developed a mouse model of septic shock to determine the capacity of Fh15 to suppress the cytokine storm and prevent the mortality rate of mice exposed to lethal doses of LPS. BALB/c mice were allotted into two experimental groups of 5 animals each, which received an IP injection with 12mg/kg body wt. of LPS from E. coli 0111: B4. One hour after LPS injection one group received a single IP injection with 100 μg Fh15 and the other group received two injections of 100 μg Fh15 two hours apart. Control groups received IP injection with LPS, Fh15 or PBS alone. Animals were monitored for mortality for 72 hrs. Blood samples were collected from all animals at baseline and by the time they were about to die or survived the LPS lethal challenge. As expected, all animals from LPS-control group succumbed between 12 to 24h following the LPS challenge. In contrast, between 40 to 100% of animals that received the Fh15 treatment survived to the LPS challenge. This notable increase in the survival rate was found associated to significant suppression in the production of pro-inflammatory cytokines such as IL-12, TNF-a and IFN-g, which were measured in serum by BioPlex.

Diagnosis of Indolent Clonorchis sinensis and Opisthorchis viverrini Infections as Risk Factors for Cholangiocarcinoma: An Unmet Medical Need.

Cholangiocarcinoma is a highly aggressive cancer of the biliary tract epithelium. This form of cancer is prevalent in Asia, and recent reports show that its incidence is relatively rare but increasing in the United States. Although risk factors for cholangiocarcinoma have yet to be elucidated, a growing body of literature suggests chronic infection of genetically susceptible individuals with the food-borne zoonotic trematodes Clonorchis sinensis (C sinensis) and Opisthorchis viverrini (O viverrini) may play a role. Although most infected people remain asymptomatic, untreated indolent infections with C sinensis and O viverrini may persist in peripheral intrahepatic bile ducts for almost 30 years. During this period, the trematodes' feeding activities and their excretory-secretory products may damage the bile duct epithelium and promote local inflammation. These pathological processes could then provoke epithelial desquamation, adenomatous hyperplasia, goblet cell metaplasia, periductal fibrosis, and granuloma formation that are conducive to the initiation and progression of cholangiocarcinoma in genetically susceptible people. The International Agency for Research on Cancer has determined that there is sufficient evidence in humans for the carcinogenicity of chronic infections with C sinensis and O viverrini. Timely serodiagnosis of indolent C sinensis and O viverrini infections is important as these parasites may be a risk factor for cholangiocarcinoma in veterans who served in Vietnam. About 774,000 living Americans served in Vietnam and there is an urgent need to develop sensitive and specific serologic assays to detect both acute and indolent infections. We posit that testing and treatment of high-risk populations could lead to earlier detection and treatment of cholangiocarcinoma, leading to improved overall survival.

Characterization of excretory/secretory products of the Taenia crassiceps cysticercus involved in the induction of regulatory T cells in vivo.

The ability to modulate the host immune response has allowed some parasites to establish themselves in the tissues of an immunocompetent organism. While some parasite excretion/secretion products (ESPs) were recently reported to induce differentiation of regulatory T cells (Tregs), their identity is not known. This work is aimed to identify and characterize ESPs of Taenia crassiceps cysticerci linked with Treg induction in vivo. ESPs were obtained from cultures of T. crassiceps cysticerci and inoculated in mice, measuring Treg levels by flow cytometry. Proteins in ESPs were analyzed by electrophoresis; then, ESPs were classified as either differential or conserved. Differentially included proteins were MS-sequenced and functionally characterized. Only 4 of 10 ESPs induced Tregs. Proteins with catalytic activity and those involved in immunological processes predominated, supporting the idea that these molecules could play an important role in the induction of Tregs.

Development of an enzyme linked immunosorbent assay using recombinant cathepsin B5 antigen for sero-surveillance of bovine tropical fasciolosis

Bovine tropical fasciolosis, caused by Fasciola gigantica, is a major parasitic disease in tropical countries responsible for significant production losses in animal husbandry practices. The disease is transmitted by the Radix sp. snails. In the early developmental stage of the parasite, the juveniles and immature flukes cause considerable damage to the liver parenchyma of the bovine host while migrating through the liver. The cathepsin (cat) B5 is a cysteine protease that is present in the excretory-secretory product of the fluke both in immature and adult stages. The early detection of fasciolosis is very critical in effective disease management. In this study, the cathepsin B5 gene from newly excysted juveniles were cloned, sequenced and analyzed. The phylogenetic analysis revealed existence of two distinct clades. The clade I includes cat B 1 to B3 whereas clade II consist of cat B4 to B7. Further, the present study was aimed to develop an enzyme linked immuno sorbent assay (ELISA) using recombinant cat B5 antigen. The developed enzyme immuno assay showed 95.3 % sensitivity and 92.4 % specificity with a cut-off of 60 % percent positive. It revealed weighted Kappa value as 0.768 (95 % CI 0.648–0.889) when compared with ELISA using native cathepsin protein. Hence, the developed assay can be exploited as a potent tool in the diagnosis and sero-surveillance of bovine tropical fasciolosis.

Immunization with excretory-secretory molecules of intestinal nematodes induces antigen-specific protective memory Th2 cell responses.

Parasitic nematodes infect more than 1 billion people in the global south. The development of effective antihelminthic vaccines is a crucial tool for their future elimination. Protective immune responses to nematodes depend on Gata3+ Th2 cells, which can also be induced by nematode-released products. Whether these nematode products induce antigen-specific long-lived memory T cells and thereby confer protection against a challenge infection is not known yet. Hence, we set out to characterize the formation of memory Th2 cells induced by immunization with Heligmosomoides polygyrus excretory-secretory (HES) products, infection-induced versus immunization-induced recall responses to a challenge infection, and whether HES-induced memory T cells show protective properties following adoptive transfer. Our results show that 8 weeks postimmunization, HES induces long-lived functional memory Th2 cells at the site of immunization in the peritoneal cavity. Following a H. polygyrus challenge infection, HES-immunized mice display MHC-II-dependent antigen-specific Th2 cytokine responses in the gut-draining lymph nodes, comparable to those induced by a prior natural infection. Moreover, adoptive transfer of sorted memory CD4+ T cells from HES-immunized donors reduces female worm fecundity following a challenge H. polygyrus infection in recipient mice, highlighting a protective role for immunization-induced memory T cells.

Clonorchis sinensis legumain promotes migration and invasion of cholangiocarcinoma cells via regulating tumor-related molecules

BackgroundClonorchis sinensis infection causes serious pathological changes in the bile duct and is highly correlated with cholangiocarcinoma. The excretory–secretory products (ESP) of C. sinensis play a critical role in the oncogenesis and progression of cholangiocarcinoma, while the components and precise mechanism remain unclear. Here, we evaluated the function of C. sinensis legumain (Cslegumain) in promoting the invasion and migration of cholangiocarcinoma cells and the mechanism involved.MethodsThe structural and molecular characteristics of Cslegumain were predicted and analyzed using the online program Phyre2. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to test the transcriptional level of Cslegumain and its localization in the adult. Native Cslegumain was detected by western blotting assay. The effects of Cslegumain on the proliferation, invasion and migration of cholangiocarcinoma cells were checked using CCK-8 assay, Matrigel transwell assay and scratch wound healing assay. Expression levels of tumor-related molecules regulated by Cslegumain were evaluated by qRT-PCR and western blotting assay.ResultsCslegumain showed high similarity with human legumain in the secondary and tertiary structures and displayed higher transcriptional levels in the adult worm than in the metacercariae. Native Cslegumain was detected in a catalytic form and was localized mainly in the intestine of the C. sinensis adult and epithelial cells of the intrahepatic bile duct. After transfection into RBE cells, Cslegumain showed high ability in promoting the invasion and migration but not the proliferation of cholangiocarcinoma RBE cells. Furthermore, the expression levels of some molecules including E-cadherin and N-cadherin were downregulated, while the levels of α-actinin 4, β-catenin and inducible nitric oxide synthase (iNOS) were upregulated.ConclusionsOur findings indicated that Cslegumain showed very similar structures as those of human legumain and could promote the invasion and migration of cholangiocarcinoma cells by regulating some tumor-related molecules.Graphical

The 22nd Chromatography Component of the Fasciola gigantica Excretory-Secretory Products Decreased the Proliferation of Peripheral Blood Mononuclear Cells from Buffalo.

The 22nd chromatography component (F22) of the Fasciola gigantica excretory-secretory products (FgESP) shows better diagnostic value than the FgESP, and diagnostic methods based on F22 have also been established. Thus, exploring its immunomodulatory function and potential as a molecular vaccine candidate is attractive. In the present study, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) in the innate immune response was preliminarily studied using the FgESP as a control. PBMCs were incubated with concanavalin A (ConA) and phytohemagglutinin (PHA) at optimal (1 µg/well) or suboptimal (0.25 µg/well) doses coupled with FgESP and F22 at different doses (1-16 µg/well). Cell proliferation was then assessed by microenzyme reaction colorimetry (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay). In addition, the components of F22 were also explored by mass spectrometry and then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to infer their functions. The results indicated that FgESP decreased the proliferation of PBMCs stimulated with ConA and PHA at specific doses, whereas F22 significantly decreased the proliferation of PBMCs stimulated with ConA and PHA at both optimal and suboptimal doses (p < 0.05). Two hundred and sixteen proteins were identified in F22, and these included 86 proteins that could be assigned to more than one pathway and some with robust immunomodulatory ability. Further studies should be performed to investigate the immunomodulatory function of F22 in the adaptive immune response, and the components of F22 can be further studied as potential vaccine candidate molecules.

Large-scale proteomic analysis of T. spiralis muscle-stage ESPs identifies a novel upstream motif for in silico prediction of secreted products

The Trichinella genus contains parasitic nematodes capable of infecting a wide range of hosts including mammals, birds and reptiles. Like other helminths, T. spiralis secretes a complex mixture of bioactive molecules capable of modulating its immediate surroundings and creating a hospitable environment for growth, survival and ultimately transmission. The constitution of these excretory-secretory products (ESPs) changes depending on the tissue niche and the specific stage of parasite development. Unique to T. spiralis is a true intracellular stage wherein larvae develop inside striated myotubes. Remarkably, the parasite larvae do not destroy the host cell but rather reprogram it to support their presence and growth. This transformation is largely mediated through stage-specific secretions released into the host cell cytoplasm. In this study, we apply state of the art proteomics and computational approaches to elucidate the composition and functions of muscle-stage T. spiralis ESPs. Moreover, we define a recurring, upstream motif associated with the stichosome, the main secretory organ of this worm, and can be used to predict secreted proteins across experimentally less tractable T. spiralis life cycle stages.

Immunomodulators secreted from parasitic helminths act on pattern recognition receptors

Excretory-secretory (ES) products from parasitic helminths contain immunomodulatory molecules, which can regulate host immune responses. These immunomodulatory molecules are crucial for successful parasitism, and play roles in tissue migration, maturation, and reproduction. Some target pattern recognition receptors (PRRs), including toll-like receptor, C-type lectin receptor, receptor for advanced glycation end products, and nucleotide-binding oligomerization domain-like receptor. PRRs trigger activation of signaling cascades, inducing innate inflammatory responses and adaptive immunity in hosts. This article reviews ES immunomodulators identified in parasitic helminths that act on PRRs, and their PRR-facilitated immune-regulatory mechanisms. In addition, we describe the therapeutic potential of ES immunomodulators for allergic and inflammatory diseases.

Excretory/Secretory Proteome of Females and Males of the Hookworm Ancylostoma ceylanicum.

The dynamic host-parasite mechanisms underlying hookworm infection establishment and maintenance in mammalian hosts remain poorly understood but are primarily mediated by hookworm's excretory/secretory products (ESPs), which have a wide spectrum of biological functions. We used ultra-high performance mass spectrometry to comprehensively profile and compare female and male ESPs from the zoonotic human hookworm Ancylostoma ceylanicum, which is a natural parasite of dogs, cats, and humans. We improved the genome annotation, decreasing the number of protein-coding genes by 49% while improving completeness from 92 to 96%. Compared to the previous genome annotation, we detected 11% and 10% more spectra in female and male ESPs, respectively, using this improved version, identifying a total of 795 ESPs (70% in both sexes, with the remaining sex-specific). Using functional databases (KEGG, GO and Interpro), common and sex-specific enriched functions were identified. Comparisons with the exclusively human-infective hookworm Necator americanus identified species-specific and conserved ESPs. This is the first study identifying ESPs from female and male A. ceylanicum. The findings provide a deeper understanding of hookworm protein functions that assure long-term host survival and facilitate future engineering of transgenic hookworms and analysis of regulatory elements mediating the high-level expression of ESPs. Furthermore, the findings expand the list of potential vaccine and diagnostic targets and identify biologics that can be explored for anti-inflammatory potential.

Protease activity in the excretory-secretory products of nymphal stage of Linguatula serrata: First detection of a metalloprotease

The aim of this study was to evaluate the presence of proteases and determine the main protease present in the excretory-secretory products (ESPs) from nymphal stage of Linguatula serrata. Infected mesenteric lymph nodes of goats were collected from Tabriz slaughterhouse, northwestern Iran. Recovered Linguatula serrata nymphs were immersed in culture medium (MEM), then ESPs were collected and protease activity in presence of specific inhibitors was assayed. Protease enzyme was further characterised by SDS-PAGE. The results of this study showed that the main protease in the ESPs from the nymphal stage of L. serrata was a metalloprotease that was resistant to heat. In conclusion, these data show that a major protease secreted by the larval stage of L. serrata exhibited properties that may play a role in the pathogenesis of L. serrata nymphs.