Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

The crosstalk between cholangiocytes and hepatic stellate cells promotes the progression of epithelial-mesenchymal transition and periductal fibrosis during Clonorchis sinensis infection.

ABSTRACT: BACKGROUND: Clonorchis sinensis infection is one of the risk factors that provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis and even cholangiocarcinoma (CCA). Disrupted or aberrant intercellular communication among liver-constituting cells leads to pathological states that cause various hepatic diseases. This study was designed to investigate the pathological changes caused by C. sinensis excretory-secretory products (ESPs) in non-cancerous human cell lines (cholangiocytes [H69 cell line] and human hepatic stellate cells [LX2 cell line]) and their intercellular crosstalk, as well the pathological changes in infected mouse liver tissues. METHODS: The cells were treated with ESPs, following which transforming growth factor beta 1 (TGF-ß1) and interleukin-6 (IL-6) secretion levels and epithelial-mesenchymal transition (EMT)- and fibrosis-related protein expression were measured. The ESP-mediated cellular motility (migration/invasion) between two cells was assessed using the Transwell and three-dimensional microfluidic assay models. The livers of C. sinensis-infected mice were stained using EMT and fibrotic marker proteins. RESULTS: Treatment of cells with ESPs increased TGF-ß1 and IL-6 secretion and the expression of EMT- and fibrosis-related proteins. The ESP-mediated mutual cell interaction further affected the cytokine secretion and protein expression levels and promoted cellular motility. N-cadherin overexpression and collagen fiber deposition were observed in the livers of C. sinensis-infected mice. CONCLUSIONS: These findings suggest that EMT and biliary fibrosis occur through intercellular communication between cholangiocytes and hepatic stellate cells during C. sinensis infection, promoting malignant transformation and advanced hepatobiliary abnormalities.

Proteomic differences between extracellular vesicles and extracellular vesicle-depleted excretory/secretory products of barber's pole worm.

BACKGROUND: Components of excretory/secretory products (ESPs) of helminths have been proposed as vaccine targets and shown to play a role in modulating host immune responses for decades. Such research interest is further increased by the discovery of extracellular vesicles (EVs) in the ESPs of parasitic worms. Although efforts have been made to reveal the cargos of EVs, little is known about the proteomic differences between EVs and canonical ESPs released by parasitic worms from animals. METHODS: The total ESPs of Haemonchus contortus (barber's pole worm) were obtained by short-term in vitro culturing of young adult worms, and small EVs were isolated from ESPs using an ultracentrifugation method. Data-dependent acquisition (DDA) label-free Nano-LC-MS/MS was used to quantify the proteomic difference between small EVs and EV-depleted ESPs of H. contortus. Functional annotation and enrichment of the differential proteins were performed regarding cellular components, molecular functions, pathways, and/or biological processes. RESULTS: A total of 1697 proteins were identified in small EVs and EV-depleted ESPs of H. contortus adult worms, with 706 unique proteins detected in the former and 597 unique proteins in the latter. It was revealed that proteins in small EVs are dominantly cytoplasmic, whereas proteins in EV-depleted ESPs are mainly extracellular; canonical ESPs such as proteases and small GTPases were abundantly detected in small EVs, and SCP/TAP-, DUF-, and GLOBIN domain-containing proteins were mainly found in EV-depleted ESPs. Compared with well-characterised proteins in small EVs, about 50% of the proteins detected in EV-depleted ESPs were poorly characterised. CONCLUSIONS: There are remarkable differences between small EVs and EV-depleted ESPs of H. contortus in terms of protein composition. Immune modulatory effects caused by nematode ESPs are possibly contributed mainly by the proteins in small EVs.

Effects of protoscoleces excretory-secretory products of Echinococcus granulosus on hepatocyte growth, function, and glucose metabolism.

Cystic echinococcosis (CE) is one of the most widespread and harmful zoonotic parasitic diseases, which most commonly affects the liver. In this study, we characterized multiple changes in mouse hepatocytes following treatment with excretory-secretory products (ESPs) of Echinococcus granulosus protoscoleces (Eg-PSCs) by a factorial experiment. The cell counting kit-8 assay (CCK-8), the 5-ethynyl-2'-deoxyuridine (EdU) assay, and flow cytometry were used to detect the growth of hepatocytes. Inverted microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used to observe the morphology and ultrastructure of hepatocytes. An automatic biochemical analyzer and an ELISA detection kit were used to determine six conventional hepatocyte enzymatic indices, the levels of five hepatocyte-synthesized substances, and the contents of glucose and lactate. Western blot analysis was conducted to analyze the protein expression of three apoptosis-related proteins, Bax, Bcl-2, cleaved caspase-3, and six glucose metabolism pathways rate-limiting enzymes in hepatocytes. The results showed that ESPs inhibited hepatocyte proliferation and promoted hepatocyte apoptosis. The cell membrane and microvilli of hepatocytes changed, and the nucleus, mitochondria and rough endoplasmic reticulum were damaged to varying degrees. The contents of iron, albumin (ALB), uric acid (UA) and urea were increased, and the activities of six enzymes in hepatocytes were increased except for the decrease of transferrin (TRF). The expression levels of all six key enzymes in the glucose metabolism pathway in hepatocytes were reduced. Our characterization provides a basis for further research on the pathogenesis, prevention and treatment of CE.

Metabolite profiling of Trichinella spiralis adult worms and muscle larvae identifies their excretory and secretory products.

Human trichinellosis is a parasitic infection caused by roundworms belonging to the genus Trichinella, especially Trichinella spiralis. Early and accurate clinical diagnoses of trichinellosis are required for efficacious prognosis and treatment. Current drug therapies are limited by antiparasitic resistance, poor absorption, and an inability to kill the encapsulating muscle-stage larvae. Therefore, reliable biomarkers and drug targets for novel diagnostic approaches and anthelmintic drugs are required. In this study, metabolite profiles of T. spiralis adult worms and muscle larvae were obtained using mass spectrometry-based metabolomics. In addition, metabolite-based biomarkers of T. spiralis excretory-secretory products and their related metabolic pathways were characterized. The metabolic profiling identified major, related metabolic pathways involving adenosine monophosphate (AMP)-dependent synthetase/ligase and glycolysis/gluconeogenesis in T. spiralis adult worms and muscle larvae, respectively. These pathways are potential drug targets for the treatment of the intestinal and muscular phases of infection. The metabolome of larva excretory-secretory products was characterized, with amino acid permease and carbohydrate kinase being identified as key metabolic pathways. Among six metabolites, decanoyl-l-carnitine and 2,3-dinor-6-keto prostaglandin F1α-d9 were identified as potential metabolite-based biomarkers that might be related to the host inflammatory processes. In summary, this study compared the relationships between the metabolic profiles of two T. spiralis growth stages. Importantly, the main metabolites and metabolic pathways identified may aid the development of novel clinical diagnostics and therapeutics for human trichinellosis and other related helminthic infections.

Excretory/secretory products from Trichinella spiralis adult worms ameliorate myocardial infarction by inducing M2 macrophage polarization in a mouse model.

BACKGROUND: Ischemia-induced inflammatory response is the main pathological mechanism of myocardial infarction (MI)-caused heart tissue injury. It has been known that helminths and worm-derived proteins are capable of modulating host immune response to suppress excessive inflammation as a survival strategy. Excretory/secretory products from Trichinella spiralis adult worms (Ts-AES) have been shown to ameliorate inflammation-related diseases. In this study, Ts-AES were used to treat mice with MI to determine its therapeutic effect on reducing MI-induced heart inflammation and the immunological mechanism involved in the treatment. METHODS: The MI model was established by the ligation of the left anterior descending coronary artery, followed by the treatment of Ts-AES by intraperitoneal injection. The therapeutic effect of Ts-AES on MI was evaluated by measuring the heart/body weight ratio, cardiac systolic and diastolic functions, histopathological change in affected heart tissue and observing the 28-day survival rate. The effect of Ts-AES on mouse macrophage polarization was determined by stimulating mouse bone marrow macrophages in vitro with Ts-AES, and the macrophage phenotype was determined by flow cytometry. The protective effect of Ts-AES-regulated macrophage polarization on hypoxic cardiomyocytes was determined by in vitro co-culturing Ts-AES-induced mouse bone marrow macrophages with hypoxic cardiomyocytes and cardiomyocyte apoptosis determined by flow cytometry. RESULTS: We observed that treatment with Ts-AES significantly improved cardiac function and ventricular remodeling, reduced pathological damage and mortality in mice with MI, associated with decreased pro-inflammatory cytokine levels, increased regulatory cytokine expression and promoted macrophage polarization from M1 to M2 type in MI mice. Ts-AES-induced M2 macrophage polarization also reduced apoptosis of hypoxic cardiomyocytes in vitro. CONCLUSIONS: Our results demonstrate that Ts-AES ameliorates MI in mice by promoting the polarization of macrophages toward the M2 type. Ts-AES is a potential pharmaceutical agent for the treatment of MI and other inflammation-related diseases.

The neuro-exocrine secretion: A new type of gland in tapeworms?

The phenomenon of exocrine secretion via nervous cells into the host tissue has been discovered in cestodes. In five cestode species of different orders specialized "cup-shaped" free nerve endings located in the tegument have been found. Their ultrastructure is characterized by the presence of a septate junction, a thin support ring and neurosecretory vesicles 90-110 nm in diameter, which are secreted onto the surface of the tegument through a thin pore. The phenomenon is referred to in this article as the neuro-exocrine secretion. We observed a direct relationship between neurosecretory processes in the deep subtegument and free endings in a series of ultrathin sections in two species. The peripheral neurosecretory neurons of species studied are characterized by similar ultrastructural features: size and location; diameter of neurosecretory granules; absence of microtubules and mitochondria in the neurites. The size of neurosecretory granules has been found to decrease from perikaryon towards neurosecretory terminals that lead to the tegument. In two species, we examined the neurosecretion during incubation in the host's blood serum. Depending on the time of incubation we have shown the changes a) in the diameter of the cup-shaped endings, b) in the number of secretory vesicles in the endings; c) changes in number and diameter of neurosecretory vesicles in the processes of neurosecretory neurons in the subtegument. The detected changes differ in D.dendriticus and L.interrupta and, taken together, indirectly confirm the secretory specialization of the cup-shaped endings. Supposed targets for the neurosecretory neurons in the studied cestodes are the following: (a) eccrine frontal gland ducts, especially their terminal regions involved in the release of secretory products; (b) longitudinal and circular muscles in the subtegument region; (c) the basal membrane of the tegument. Besides the discovered secretion vesicles through the cup-shaped terminals, we observed vacuoles derived from the basal membrane of the tegument containing extracellular substances released into the host tissue. Their possible role in the release of neurosecretory substances is discussed. Considering the data acquired via immunocytochemical methods, an assumption about involvement of FMRFamide-like related peptides (FaRPs) in the neuro-exocrine secretion is proposed. Possible functions of the neuro-exocrine secretion are discussed in the context of host-parasite interactions.

Transcriptome profiling of A549 non-small cell lung cancer cells in response to Trichinella spiralis muscle larvae excretory/secretory products.

Trichinella spiralis (T. spiralis) muscle-larva excretory/secretory products (ML-ESPs) is a complex array of proteins with antitumor activity. We previously demonstrated that ML-ESPs inhibit the proliferation of A549 non-small cell lung cancer (NSCLC) cell line. However, the mechanism of ML-ESPs against A549 cells, especially on the transcriptional level, remains unknow. In this study, we systematically investigated a global profile bioinformatics analysis of transcriptional response of A549 cells treated with ML-ESPs. And then, we further explored the transcriptional regulation of genes related to glucose metabolism in A549 cells by ML-ESPs. The results showed that ML-ESPs altered the expression of 2,860 genes (1,634 upregulated and 1,226 downregulated). GO and KEGG analysis demonstrated that differentially expressed genes (DEGs) were mainly associated with pathway in cancer and metabolic process. The downregulated genes interaction network of metabolic process is mainly associated with glucose metabolism. Furthermore, the expression of phosphofructokinase muscle (PFKM), phosphofructokinase liver (PFKL), enolase 2 (ENO2), lactate dehydrogenase B (LDHB), 6-phosphogluconolactonase (6PGL), ribulose-phosphate-3-epimerase (PRE), transketolase (TKT), transaldolase 1 (TALDO1), which genes mainly regulate glycolysis and pentose phosphate pathway (PPP), were suppressed by ML-ESPs. Interestingly, tricarboxylic acid cycle (TCA)-related genes, such as pyruvate dehydrogenase phosphatase 1 (PDP1), PDP2, aconitate hydratase 1 (ACO1) and oxoglutarate dehydrogenase (OGDH) were upregulated by ML-ESPs. In summary, the transcriptome profiling of A549 cells were significantly altered by ML-ESPs. And we also provide new insight into how ML-ESPs induced a transcriptional reprogramming of glucose metabolism-related genes in A549 cells.

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

BACKGROUND: 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. METHODS: Here, we examined the therapeutic effect of TSIIA by real-time qPCR, western blotting, activity assay, and cell viability assays. RESULTS: 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. CONCLUSION: 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.

CD4+ T Cell Epitope Identification from Complex Parasite Antigen Mixtures.

Antigen complexity represents a major challenge for scoring CD4+ T cell immunogenicity, a key hallmark of immunity and with great potential to improve vaccine development. In this chapter, we provide a comprehensive picture of a pipeline that can be applied to virtually any complex antigen to overcome different limitations. Antigens are characterized by Mass Spectrometry to determine the available protein sources and their abundances. A reconstituted in vitro antigen processing system is applied along with bioinformatics tools to prioritize the list of candidates. Finally, the immunogenicity of candidate peptides is validated ex vivo using PBMCs from HLA-typed individuals. This protocol compiles the essential information for executing the whole pipeline while focusing on the candidate epitope prioritizing scheme.

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.

Potential roles of Toxocara canis larval excretory secretory molecules in immunomodulation and immune evasion.

Toxocara canis larvae invade various tissues of different vertebrate species without developing into adults in paratenic host. The long-term survival of the larvae despite exposure to the well-armed immune response is a notable achievement. The larvae modulate the immune response to help the survival of both the host and the larvae. They skew the immune response to type 2/regulatory phenotype. The outstanding ability of the larvae to modulate the host immune response and to evade the immune arms is attributed to the secretion of Toxocara excretory-secretory products (TESPs). TESPs are complex mixture of differing molecules. The present review deals with the molecular composition of the TESPs, their interaction with the host molecules, their effect on the innate immune response, the receptor recognition, the downstream signals the adaptive immunity and the repair of tissues. This review also addresses the role of TESPs molecules in the immune evasion strategy and the potential effect of the induced immunomodulation in some diseases. Identification of parasite components that influence the nematode-host interactions could enhance understanding the molecular basis of nematode pathogenicity. Furthermore, the identification of helminths molecules with immunomodulatory potential could be used in immunotherapies for some diseases.

Ascaris suum excretory/secretory products differentially modulate porcine dendritic cell subsets.

Helminths produce excretory/secretory products (E/S) which can modulate the immune responses of their hosts. Dendritic cells (DC) are essential for initiating the host T cell response and are thus potential targets for modulation by helminth E/S. Here we study immunomodulation of porcine peripheral blood DC subsets following ex vivo stimulation with E/S from Ascaris suum, a common helminth of pigs with considerable public health and economic importance. Our data showed that the relative frequencies of DC subsets in porcine blood differ, with plasmacytoid DC (pDC) being the most prominent in healthy 6-month-old pigs. pDC are an important cytokine source, and we found that A. suum E/S suppressed production of the type 1 cytokines IL-12p40 and TNF-α by this subset following toll-like receptor (TLR) ligation. In contrast, conventional DC (cDC) are more efficient antigen presenters, and the expression of CD80/86, costimulatory molecules essential for efficient antigen presentation, were modulated differentially by A. suum E/S between cDC subsets. CD80/86 expression by type 1 cDC (cDC1) following TLR ligation was greatly suppressed by the addition of A. suum E/S, while CD80/86 expression by type 2 cDC (cDC2) was upregulated by A. suum E/S. Further, we found that IFN-γ production by natural killer (NK) cells following IL-12 and IL-18 stimulation was suppressed by A. suum E/S. Finally, in the presence of E/S, IFN-γ production by CD4+ T cells co-cultured with autologous blood-derived DC was significantly impaired. Together, these data provide a coherent picture regarding the regulation of type 1 responses by A. suum E/S. Responsiveness of pDC and cDC1 to microbial ligands is reduced in the presence of E/S, effector functions of Th1 cells are impaired, and cytokine-driven IFN-γ release by NK cells is limited.

Excretory-Secretory Products of Trichomonas vaginalis Cause Apoptosis in Mouse Sperm in Vitro.

Excretory-secretory products (ESP) of T. vaginalis have been shown to inhibit sperm motility, viability, and functional integrity, leading to a decreased fertilization rate in vitro. This study investigated whether T. vaginalis induce apoptosis and ultrastructural changes of sperm using flow cytometry and electron microscopy. Incubation of sperm with T. vaginalis ESP increased phosphatidylserine externalization and DNA fragmentation, and decreased mitochondrial membrane potential. Transmission electron microscopy of sperm incubated with ESP revealed abnormal features such as distorted heads, broken necks, and acrosomes exocytosis. This is the first report that demonstrates a direct impact of T. vaginalis ESP on sperm apoptosis and architecture in vitro.

A ShK-like Domain from Steinernema carpocapsae with Bioinsecticidal Potential.

Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per adult within 24 h. Oral administration of the fusion protein significantly reduced the locomotor activity of insects after 48 h (p < 0.05, Tukey's test). These data show that this nematode expresses insecticidal peptides with potential as next-generation insecticides.

Two Distinct Superoxidase Dismutases (SOD) Secreted by the Helminth Parasite Fasciola hepatica Play Roles in Defence against Metabolic and Host Immune Cell-Derived Reactive Oxygen Species (ROS) during Growth and Development.

The antioxidant superoxide dismutase (SOD) catalyses the dismutation of superoxide, a dangerous oxygen free radical, into hydrogen peroxide and molecular oxygen. Superoxide generation during the oxidative burst of the innate immune system is considered a key component of the host defence against invading pathogens. We demonstrate the presence and differential expression of two SODs in Fasciola hepatica, a leaderless cytosolic (FhSOD1) and an extracellular (FhSOD3) form containing a secretory signal peptide, suggesting that the parasites exploit these enzymes in distinct ways to counteract reactive oxygen species (ROS) produced by cellular metabolism and immune defences. Both enzymes are highly expressed by the infective newly excysted juvenile (NEJ) stages and are found in abundance in their excretory-secretory products (ES), but only FhSOD1 is present in adult ES, suggesting that the antioxidants have different functions and pathways of secretion, and are under separate temporal expression control during the migration, growth, and development of the parasite. Functionally, the recombinant FhSOD1 and FhSOD3 exhibit similar activity against superoxide to their mammalian counterparts. Confocal immuno-localisation studies demonstrated the presence of FhSOD1 and FhSOD3 on the NEJ tegument and parenchyma, supporting our suggestion that these enzymes are secreted during host invasion to protect the parasites from the harmful oxidative bursts produced by the activated innate immune response. By producing superoxide enzymatically in vitro, we were able to demonstrate robust killing of F. hepatica NEJ within 24 h post-excystment, and that the lethal effect of ROS was nullified with the addition of SOD and catalase (the antioxidant enzyme responsible for the dismutation of hydrogen peroxide, a by-product of the SOD reaction). This study further elucidates the mechanism by which F. hepatica protects against ROS derived from cellular metabolism and how the parasite could mitigate damage caused by the host's immune response to benefit its survival.

Excretory-secretory products from the brown stomach worm, Teladorsagia circumcincta, exert antimicrobial activity in in vitro growth assays.

BACKGROUND: Over the past decade, evidence has emerged of the ability of gastrointestinal (GI) helminth parasites to alter the composition of the host gut microbiome; however, the mechanism(s) underpinning such interactions remain unclear. In the current study, we (i) undertake proteomic analyses of the excretory-secretory products (ESPs), including secreted extracellular vesicles (EVs), of the 'brown stomach worm' Teladorsagia circumcincta, one of the major agents causing parasite gastroenteritis in temperate areas worldwide; (ii) conduct bioinformatic analyses to identify and characterise antimicrobial peptides (AMPs) with putative antimicrobial activity; and (iii) assess the bactericidal and/or bacteriostatic properties of T. circumcincta EVs, and whole and EV-depleted ESPs, using bacterial growth inhibition assays. METHODS: Size-exclusion chromatography was applied to the isolation of EVs from whole T. circumcincta ESPs, followed by EV characterisation via nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of EVs and EV-depleted ESPs was conducted using liquid chromatography-tandem mass spectrometry, and prediction of putative AMPs was performed using available online tools. The antimicrobial activities of T. circumcincta EVs and of whole and EV-depleted ESPs against Escherichia coli were evaluated using bacterial growth inhibition assays. RESULTS: Several molecules with putative antimicrobial activity were identified in both EVs and EV-depleted ESPs from adult T. circumcincta. Whilst exposure of E. coli to whole ESPs resulted in a significant reduction of colony-forming units over 3 h, bacterial growth was not reduced following exposure to worm EVs or EV-depleted ESPs. CONCLUSIONS: Our data points towards a bactericidal and/or bacteriostatic function of T. circumcincta ESPs, likely mediated by molecules with antimicrobial activity.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis.

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specific than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specific (a probably arginine kinase of 37 kDa), while other antigenic bands were highly sensitive but low specific. Proteomic analysis revealed differences between the protein profiles from E/S-RPMI and E/S-PBS since only one-third of all proteins identified were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identified as plasminogen-binding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

Parasitic helminths and the host microbiome - a missing 'extracellular vesicle-sized' link?

Infections by gastrointestinal (GI) helminths have been associated with significant alterations of the structure of microbial communities inhabiting the host gut. However, current understanding of the biological mechanisms that regulate these relationships is still lacking. We propose that helminth-derived extracellular vesicles (EVs) likely represent key players in helminth-microbiota crosstalk. Here, we explore knowledge of helminth EVs with an emphasis on their putative antimicrobial properties, and we argue that (i) an enhanced understanding of the mechanisms governing such interactions might assist the discovery and development of novel strategies of parasite control, and that (ii) the identification and characterisation of helminth molecules with antimicrobial properties might pave the way towards the discovery of novel antibiotics, thus aiding the global fight against antimicrobial resistance.

Serological testing for Trichinella infection in animals and man: Current status and opportunities for advancements.

Serological tests are widely used for the detection of Trichinella spp. infections in animals and humans. Despite some limitations, (such as low sensitivity in the early period after infection) ELISA and western blot testing have demonstrated good performance when excretory/secretory products from muscle larvae are used as antigens in agreement with the International Commission on Trichinellosis. Over recent decades, considerable progress has been made in the characterization of Trichinella-derived molecules in the hope of improving diagnosis, mainly during the early days post infection. Despite these efforts, validated tests using characterized antigens for early diagnosis are still not available. However, combining currently available sero-diagnostic tools with clinical and epidemiological data provides valuable information on Trichinella infections in humans and animals as shown in this review.