Excretory-secretory Molecules Research Articles

The production of excretory-secretory molecules from Heligmosomoides polygyrus bakeri fourth stage larvae varies between mixed and single sex cultures

BackgroundExcretory-secretory (ES) products are crucial in maintaining helminths in the host. Consequently, the proteins of ES are potential vaccine molecules and potential therapeutic agents for autoimmune diseases. Heligmosomoides polygyrus bakeri, a gastrointestinal parasite of mice, is a model of hookworm infection in humans. ES produced by both sexes of H. polygyrus bakeri L4 stage cultured separately shows different immunomodulatory properties than ES obtained when both sexes are cultured together. Accordingly, the objective of this study was to identify and compare the excretory-secretory molecules from single-sex and mixed cultures.MethodsThe composition of ES of male and female L4 stage nematodes in the presence (cultured together) or absence (cultured alone) of the opposite sex was examined. Proteins were identified using mass spectrometry. The functions of identified proteins were explored with Blast2GO.ResultsA total of 258 proteins derived from mixed larval culture in the presence of sex pheromones were identified, 160 proteins from pure female cultures and 172 from pure male cultures. Exposure of nematodes to the sex pheromones results in abundant production of proteins with immunomodulatory properties such as Val proteins, acetylcholinesterases, TGF-β mimic 9 and HpARI. Proteins found only in ES from mixed larval cultures were TGF-β mimics 6 and 7 as well as galectin.ConclusionsThe presence of the opposite sex strongly influences the composition of ES products, probably by chemical (pheromone) communication between individuals. However, examination of the composition of ES from various conditions gives an opportunity for searching for new potentially therapeutic compounds and anthelminthics as well as components of vaccines. Manipulation of the nematode environment might be important for the studies on the immunomodulatory potential of nematodes.Graphical

Cellular and humoral peritoneal immunity to Mesocestoides vogae metacestode infection in mice

BackgroundHere, Mesocestoides (M.) vogae infection in mice is proposed as a suitable experimental model for studying the immunity in the peritoneal cavity of mice.MethodsTo investigate the kinetics of immune parameters in M. vogae-infected mice, we detected, using flow cytometry, the expression of selected lymphoid and myeloid markers within the peritoneal cell population at day 0, 3, 6, 10, 14, 19, 25, 30 and 35 post-infection. Then, using ELISA, we analyzed the cytokine IFN-γ, TGF-β, IL-4 and IL-10 responses and the levels of anti-M. vogae IgG and IgM antibodies in the peritoneal lavage fluid. Cells isolated from the peritoneal cavity were subjected to further molecular analysis. To assess cell activation, peritoneal cells were exposed to LPS, and culture supernatants were collected and assayed for the level of cytokines and production of nitrite. Ly6C+ and Ly6G+ cells were isolated using MACS from the peritoneal cells at day 35 post-infection. Both MACS-isolated subsets were co-cultured with preactivated T cells to measure their suppressive capacity. Next, the role of parasite excretory-secretory antigens in induction of CD11b+ myeloid cells with the suppressive phenotype and the production of IL-10 was examined.ResultsIn the peritoneal cavity an initial increase of CD11b+Gr-1+F4/80highMHC IIhigh cells, NK, NKT cells and CD8+ cytotoxic T cells was observed in the first week of infection. At day 14 post-infection, an increase in the number of myeloid CD11b+Gr-1+ cells was detected, and most of this cell population expressed low levels of F4/80 and MHC II in later stages of infection, suggesting the impairment of antigen-presenting cell functions, probably through the excretory-secretory molecules. Moreover, we confirmed that peritoneal Gr1+ cells (Ly6C+ and Ly6G+ population) are phenotypically and functionally consistent with myeloid-derived suppressor cells. Metacestode infection elicited high levels of IL-10 and upregulated STAT-3 in peritoneal cells. A higher level of IgM suggests that this isotype may be predominant and is involved in the host protection.ConclusionsMesocestoides vogae tetrathyridia induced the recruitment of immunosuppressive cell subsets, which may play a key role in the downregulation of immune response in long-term parasitic diseases, and excretory-secretory antigens seem to be the main regulatory factor. Graphical

The somatic proteins of Toxocara canis larvae and excretory-secretory products revealed by proteomics

Toxocariasis is a widespread helminth infection of dogs and cats, caused by Toxocara canis and Toxocara cati larvae, respectively. Toxocara spp. can cause zoonotic infections in humans by invading tissues and organs causing pathology. Toxocara spp. larvae release excretory-secretory molecules (TES) into the body of their host that are fundamental to the host-parasite interaction and could be used as targets for novel diagnostics and vaccines. In the present study, we identified 646 T. canis proteins from TES and larval extract using 1D-SDS PAGE followed by mass spectrometry. A wide range of proteins was identified that may play a role both in the induction of the host immune response and host pathology, and in parasite metabolism and survival. Among these proteins there are potential candidates for novel diagnostics and vaccines for dogs and cats toxocariases.

Lipid binding proteins from parasitic platyhelminthes

Two main families of lipid binding proteins have been identified in parasitic Platyhelminthes: hydrophobic ligand binding proteins (HLBPs) and fatty acid binding proteins (FABPs). Members of the former family of proteins are specific to the Cestoda class, while FABPs are conserved across a wide range of animal species. Because Platyhelminthes are unable to synthesize their own lipids, these lipid-binding proteins are important molecules in these organisms. HLBPs are a high molecular mass complex of proteins and lipids. They are composed of subunits of low molecular mass proteins and a wide array of lipid molecules ranging from CoA esters to cholesterol. These proteins are excretory-secretory molecules and are key serological tools for diagnosis of diseases caused by cestodes. FABPs are mainly intracellular proteins of low molecular weight. They are also vaccine candidates. Despite that the knowledge of their function is scarce, the differences in their molecular organization, ligand preferences, intra/extracellular localization, evolution, and phylogenetic distribution, suggest that platyhelminths HLBPs and FABPs should play different functions. FABPs might be involved in the removal of fatty acids from the inner surface of the cell membrane and in their subsequent targeting to specific cellular destinations. In contrast, HLBPs might be involved in fatty acid uptake from the host environment.

Fluorescent Imaging of Antigen Released by a Skin-Invading Helminth Reveals Differential Uptake and Activation Profiles by Antigen Presenting Cells

Infection of the mammalian host by the parasitic helminth Schistosoma mansoni is accompanied by the release of excretory/secretory molecules (ES) from cercariae which aid penetration of the skin. These ES molecules are potent stimulants of innate immune cells leading to activation of acquired immunity. At present however, it is not known which cells take up parasite antigen, nor its intracellular fate. Here, we develop a technique to label live infectious cercariae which permits the imaging of released antigens into macrophages (MΦ) and dendritic cells (DCs) both in vitro and in vivo. The amine reactive tracer CFDA-SE was used to efficiently label the acetabular gland contents of cercariae which are released upon skin penetration. These ES products, termed ‘0-3hRP’, were phagocytosed by MHC-II+ cells in a Ca+ and actin-dependent manner. Imaging of a labelled cercaria as it penetrates the host skin over 2 hours reveals the progressive release of ES material. Recovery of cells from the skin shows that CFDA-SE labelled ES was initially (3 hrs) taken up by Gr1+MHC-II− neutrophils, followed (24 hrs) by skin-derived F4/80+MHC-IIlo MΦ and CD11c+ MHC-IIhi DC. Subsequently (48 hrs), MΦ and DC positive for CFDA-SE were detected in the skin-draining lymph nodes reflecting the time taken for antigen-laden cells to reach sites of immune priming. Comparison of in vitro-derived MΦ and DC revealed that MΦ were slower to process 0-3hRP, released higher quantities of IL-10, and expressed a greater quantity of arginase-1 transcript. Combined, our observations on differential uptake of cercarial ES by MΦ and DC suggest the development of a dynamic but ultimately balanced response that can be potentially pushed towards immune priming (via DC) or immune regulation (via MΦ).

Immunization of goldfish with excretory/secretory molecules of Trypanosoma danilewskyi confers protection against infection

Trypanosoma danilewskyi is a protozoan that lives in blood and other tissues of fish. In the aquaculture industry, economic losses may be substantial, since the prevalence of infection may approach 100% and the parasite may cause significant mortality in farmed carp. Most of the surviving fish acquire resistance after elimination of the primary infection. In this study, we examined whether protection against infection could be induced in naı̈ve goldfish immunized with excretory–secretory (ES) products of the parasite. The ES extracts were administered in conjunction with Freund's complete or incomplete adjuvant (FCA and FIA, respectively). Parameters used to assess the efficacy of immunization after challenge infection, included prevalence of infection, abundance of parasites, and presence of parasite-specific antibodies. Intraperitoneal inoculation of ES products in FCA conferred protection against T. danilewskyi infection ( P<0.05). Administration of ES products (with or without FIA) conferred insignificant levels of protection. In an attempt to identify the immunogenic ES molecules, we assessed whether anti-parasite antibodies present in the serum collected from experimentally infected fish or rabbits immunized with ES recognized parasite ES antigens. An immunoblot using rabbit anti-parasite antibody revealed a recognition profile of molecules (two antigens of approximately 102–104 and 70–72 kDa) similar to that of immune goldfish serum. While the antigens that confer protection need further molecular characterization, our results suggest that the administration of ES products may allow for the design of control strategies for T. danilewskyi.

Partial Characterization of the Epitope on Excretory-Secretory Products of Fasciola hepatica Recognized by Monoclonal Antibody ES78

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

Antigens from the surface and excretions/secretions of the filariform larva of Strongyloides stercoralis

The surface and excretory/secretory (ES) antigens of the infective, filariform larva (L3) of Strongyloides stercoralis were identified. These studies provide a basis for the purification of these proteins as diagnostic allergens for human strongyloidiasis. The M r values of the surface and ES molecules were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, fluorography, or silver staining following the recovery of these molecules after the radiolabelling of living parasites. At least 10 surface proteins were radioiodinated extrinsically using chloroglycoluril as the catalyst for iodination, and then extracted with detergents and/or β-mercaptoethanol. Several surface molecules of the L3 were immunogenic in humans, as determined by immunoprecipitation with sera (IHS) from infected patients. About 30 proteins were present in the ES preparation. Many ES antigens were labelled biosynthetically during the culture of larvae in media supplemented with either [ 35S]methionine or [ 14C]glucose. Furthermore, several of the surface proteins of the L3 were found with the ES antigens recoverable by culturing larvae in vitro. About 10 of the ES proteins were immunogenic as determined by immunoaffinity chromatography using IHS; and two of these antigens with M r 50 000 and 90 000 incorporated [ 35S]methionine during culture of larvae. Moreover, some ES proteins were allergenic when tested in an in vitro assay of histamine release from basophils from infected humans or monkeys. The isotype of the homocytophilic antibodies involved in this immediate hypersensitivity assay, which is the basis of a diagnostic skin test for human strongyloidiasis, appears to be IgE.

Biochemical properties of larval excretory-secretory glycoproteins of the parasitic nematode Toxocara canis

Toxocara canis larvae, infective to Man, secrete a number of antigenic macromolecules into culture medium over prolonged periods of time. These antigens have been collected and characterised with respect to molecular weight by sodium dodecyl sulphate-polyacrylamide gel electrophoresis following extrinsic and intrinsic radiolabelling, or electrophoresis followed by gel staining or immunoblotting. Panels of enzymes and lectins have been applied to examine protease sensitivity and carbohydrate composition, respectively, and a number of other biochemical data have been noted. Taken together, the excretory-secretory molecules contain more than 40% carbohydrate of which the majority is N-acetylgalactosamine and galactose. The individual antigens may readily be separated by gel filtration on a Sepharose 6B column, and it is shown that the major excretory-secretory macromolecules are all glycoproteins which differ in essential characteristics. For example, the 32 kDa antigen (TES-32) binds concanavalin A, is sensitive to a range of proteases and is the band most readily stained by silver and Coomassie blue. Both TES-120 and TES-400 components are resistant to tryptic or peptic cleavage, bind to Helix pomatia lectin and stain with periodic acid-Schiff, yet unlike TES-120, TES-400 does not incorporate radioactive methionine nor can it be stained by silver stain techniques. Finally, one protease, staphylococcal V8, reveals cleavage sites only in the TES-70 and TES-400 molecules.