A phosphorylcholine-containing glycolipid-like antigen present on the surface of infective stage larvae of Ascaris spp. is a major antibody target in infected pigs and humans

BACKGROUND: The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential. METHODOLOGY/PRINCIPAL FINDINGS: Pigs were immunized by trickle infection for 30 weeks, challenged with 2,000 eggs at week 32 and euthanized two weeks after challenge. At necropsy, there was a 100% reduction in worms recovered from the intestine and a 97.2% reduction in liver white spots in comparison with challenged non-immune control animals. Antibodies purified from the intestinal mucus or from the supernatant of cultured antibody secreting cells from mesenteric lymph nodes of immune pigs were used to probe L3 extracts to identify antibody targets. This resulted in the recognition of a 12kDa antigen (As12) that is actively shed from infective Ascaris L3. As12 was characterized as a phosphorylcholine-containing glycolipid-like antigen that is highly resistant to different enzymatic and chemical treatments. Vaccinating pigs with an As12 fraction did not induce protective immunity to challenge infection. However, serological analysis using sera or plasma from experimentally infected pigs or naturally infected humans demonstrated that the As12 ELISA was able to detect long-term exposure to Ascaris with a high diagnostic sensitivity (98.4% and 92%, respectively) and specificity (95.5% and 90.0%) in pigs and humans, respectively. CONCLUSIONS/SIGNIFICANCE: These findings show the presence of a highly stage specific, glycolipid-like component (As12) that is actively secreted by infectious Ascaris larvae and which acts as a major antibody target in infected humans and pigs

Rethinking Schistosomiasis Vaccine Development:Synthetic Vesicles

There is currently no vaccine against schistosomiasis. With few Schistosoma vaccine candidates in clinical trials, unexplored antigens from the vulnerable schistosomulum should be considered as possible vaccine candidates. In addition, we suggest developing synthetic vesicles as a new delivery vehicle and adjuvant for immunoprophylactic schistosomula vaccine candidates

Community rates of IgG4 antibodies to Ascaris haemoglobin reflect changes in community egg loads following mass drug administration

BACKGROUND:Conventional diagnostic methods for human ascariasis are based on the detection of Ascaris lumbricoides eggs in stool samples. However, studies of ascariasis in pigs have shown that the prevalence and the number of eggs detected in the stool do not correlate well with exposure of the herd to the parasite. On the other hand, an ELISA test measuring antibodies to Ascaris suum haemoglobin (AsHb) has been shown to be useful for estimating transmission intensity on pig farms. In this study, we further characterized the AsHb antigen and screened samples from a population-based study conducted in an area that is endemic for Ascaris lumbricoides in Indonesia to assess changes in AsHb antibody rates and levels in humans following mass drug administration (MDA). METHODOLOGY/PRINCIPAL FINDINGS:We developed and evaluated an ELISA to detect human IgG4 antibodies to AsHb. We tested 1066 plasma samples collected at different times from 599 subjects who lived in a village in rural Indonesia that was highly endemic for ascariasis. The community received 6 rounds of MDA for lymphatic filariasis with albendazole plus diethylcarbamazine between 2002 and 2007. While the AsHb antibody assay was not sensitive for detecting all individuals with Ascaris eggs in their stools, the percentage of seropositive individuals decreased rapidly following MDA. Reductions in antibody rates reflected decreased mean egg output per person both at the community level and in different age groups. Two years after the last round of MDA the community egg output and antibody prevalence rate were reduced by 81.6% and 78.9% respectively compared to baseline levels. CONCLUSION/SIGNIFICANCE:IgG4 antibody levels to AsHb appear to reflect recent exposure to Ascaris. The antibody prevalence rate may be a useful indicator for Ascaris transmission intensity in communities that can be used to assess the impact of control measures on the force of transmission

Novel O-linked methylated glycan antigens decorate secreted immunodominant glycoproteins from the intestinal nematode Heligmosomoides polygyrus

Glycan molecules from helminth parasites have been associated with diverse biological functions ranging from interactions with neighbouring host cell populations to down-modulation of specific host immunity. Glycoproteins secreted by the intestinal nematode Heligmosomoides polygyrus are of particular interest as the excretory-secretory products (termed HES) of this parasite contain both heat-labile and heat-stable components with immunomodulatory effects. We used MALDI-TOF-MS and LC-MS/MS to analyse the repertoire of N- and O-linked glycans released from Heligmosomoides polygyrus excretory-secretory products by PNGase A and F, β-elimination and hydrazinolysis revealing a broad range of structures including novel methylhexose- and methylfucose-containing glycans. Monoclonal antibodies to two immunodominant glycans of H. polygyrus, previously designated Glycans A and B, were found to react by glycan array analysis to a methyl-hexose-rich fraction and to a sulphated LacDiNAc (LDN; GalNAcβ1-4GlcNAc) structure, respectively. We also analysed the glycan repertoire of a major glycoprotein in Heligmosomoides polygyrus excretory-secretory products, VAL-2, which contains many glycan structures present in Heligmosomoides polygyrus excretory-secretory products including Glycan A. However, it was found that this set of glycans is not responsible for the heat-stable immunomodulatory properties of Heligmosomoides polygyrus excretory-secretory products, as revealed by the inability of VAL-2 to inhibit allergic lung inflammation. Taken together, these studies reveal that H. polygyrus secretes a diverse range of antigenic glycoconjugates, and provides a framework to explore the biological and immunomodulatory roles they may play within the mammalian host

Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans

Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed

Golgi targeting of Drosophila melanogaster β4GalNAcTB requires a DHHC protein family–related protein as a pilot

Drosophila melanogaster β4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcβ1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated β4GalNAcTB together with β4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive β4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of β4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with β4GalNAcTB and, when expressed with an ER retention signal, holds active β4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs β4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of β4GalNAcTB

E-cadherin expression in macrophages dampens their inflammatory responsiveness in vitro, but does not modulate M2-regulated pathologies in vivo

IL-4/IL-13-induced alternatively activated macrophages (M(IL-4/IL-13), AAMs or M2) are known to express E-cadherin, enabling them to engage in heterotypic cellular interactions and IL-4-driven macrophage fusion in vitro. Here we show that E-cadherin overexpression in Raw 264.7 macrophages inhibits their inflammatory response to LPS stimulation, as demonstrated by a reduced secretion of inflammatory mediators like interleukin (IL)-6, tumor necrosis factor (TNF) and nitric oxide (NO). To study the function of E-cadherin in M(IL-4/IL-13) macrophages in vivo, we generated macrophage-specific E-cadherin-deficient C57BL/6 mice. Using this new tool, we analyzed immunological parameters during two typical AAM-associated Th2-driven diseases and assessed Th2-associated granuloma formation. Although E-cadherin is strongly induced in AAMs during Taenia crassiceps helminth infections and allergic airway inflammation, its deletion in macrophages does not affect the course of both Th2 cytokine-driven diseases. Moreover, macrophage E-cadherin expression is largely redundant for granuloma formation around Schistosoma mansoni ova. Overall, we conclude that E-cadherin is a valuable AAM marker which suppresses the inflammatory response when overexpressed. Yet E-cadherin deletion in macrophages does not affect M(LPS+IFN gamma) and M(IL-4) polarization in vitro, nor in vivo macrophage function, at least in the conditions tested

Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni

Background: Penetration of skin, migration through tissues and establishment of long-lived intravascular partners require Schistosoma parasites to successfully manipulate definitive host defences. While previous studies of larval schistosomula have postulated a function for excreted/secreted (E/S) products in initiating these host-modulatory events, the role of extracellular vesicles (EVs) has yet to be considered. Here, using preparatory ultracentrifugation as well as methodologies to globally analyse both proteins and small non-coding RNAs (sncRNAs), we conducted the first characterization of Schistosoma mansoni schistosomula EVs and their potential host-regulatory cargos. Results: Transmission electron microscopy analysis of EVs isolated from schistosomula in vitro cultures revealed the presence of numerous, 30–100 nm sized exosome-like vesicles. Proteomic analysis of these vesicles revealed a core set of 109 proteins, including homologs to those previously found enriched in other eukaryotic EVs, as well as hypothetical proteins of high abundance and currently unknown function. Characterization of E/S sncRNAs found within and outside of schistosomula EVs additionally identified the presence of potential gene-regulatory miRNAs (35 known and 170 potentially novel miRNAs) and tRNA-derived small RNAs (tsRNAs; nineteen 5′ tsRNAs and fourteen 3′ tsRNAs). Conclusions: The identification of S. mansoni EVs and the combinatorial protein/sncRNA characterization of their cargo signifies that an important new participant in the complex biology underpinning schistosome/host interactions has now been discovered. Further work defining the role of these schistosomula EVs and the function/stability of intra- and extra-vesicular sncRNA components presents tremendous opportunities for developing novel schistosomiasis diagnostics or interventions

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

AbstractThe Schistosoma mansoni venom allergen-like (SmVAL) protein family consists of 29 members, each possessing a conserved α-β-α sandwich tertiary feature called the Sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain. While the SmVALs have been found in both excretory/secretory (E/S) products and in intra/sub-tegumental (non-E/S) fractions, the role(s) of this family in host/parasite relationships or schistosome developmental processes remains poorly resolved. In order to begin quantifying SmVAL functional diversity or redundancy, dissecting the specific activity (ies) of individual family members is necessary. Towards this end, we present the characterisation of SmVAL9; a protein previously found enriched in both miracidia/sporocyst larval transformation proteins and in egg secretions. While our study confirms that SmVAL9 is indeed found in soluble egg products and miracidia/sporocyst larval transformation proteins, we find it to be maximally transcribed/translated in miracidia and subsequently down-regulated during in vitro sporocyst development. SmVAL9 localisation within sporocysts appears concentrated in parenchymal cells/vesicles as well as associated with larval germinal cells. Furthermore, we demonstrate that egg-derived SmVAL9 carries an N-linked glycan containing a schistosome-specific difucosyl element and is an immunogenic target during chronic murine schistosomiasis. Finally, we demonstrate that recombinant SmVAL9 affects the expression of extracellular matrix, remodelling matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) gene products in both Biomphalaria glabrata embryonic cell (BgMMP1) and Mus musculus bone marrow-derived macrophage (MmMMP2, MmMMP9, MmMMP12, MmMMP13, MmMMP14, MmMMP28, TIMP1 and TIMP2) in vitro cultures. These findings importantly suggest that excreted/secreted SmVAL9 participates in tissue reorganisation/extracellular matrix remodelling during intra-mammalian egg translocation, miracidia infection and intra-molluscan sporocyst development/migration

Functional characterization of Schistosoma mansoni fucosyltransferases in Nicotiana benthamiana plants

Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host–parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines