Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1–4(Fucα1–3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1–4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1–3(Galβ1–6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.
BackgroundSchistosomiasis (bilharzia) is a chronic and potentially deadly parasitic disease that affects millions of people in (sub)tropical areas. An important partial immunity to Schistosoma infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during Schistosoma infection are directed against the numerous glycans expressed by Schistosoma. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown.Methodology/Principal FindingsThe binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of S. mansoni and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection.Conclusions/SignificanceUsing the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in S. mansoni infections. These findings are significant for further exploration of Schistosoma glycan antigens in relation to immunity.
In schistosomiasis, the majority of symptoms of the disease is caused by the eggs that are trapped in the liver. These eggs elicit an immune reaction that leads to the formation of granulomas. The eggshell, which is a rigid insoluble structure built from cross-linked proteins, is the site of direct interaction between the egg and the immune system. However, the exact protein composition of the insoluble eggshell was previously unknown. To identify the proteins of the eggshell of Schistosoma mansoni we performed LC-MS/MS analysis, immunostaining and amino acid analysis on eggshell fragments. For this, eggshell protein skeleton was prepared by thoroughly cleaning eggshells in a four-step stripping procedure of increasing strength including urea and SDS to remove all material that is not covalently linked to the eggshell itself, but is part of the inside of the egg, such as Reynold's layer, von Lichtenberg's envelope and the miracidium. We identified 45 proteins of which the majority are non-structural proteins and non-specific for eggs, but are house-keeping proteins that are present in large quantities in worms and miracidia. Some of these proteins are known to be immunogenic, such as HSP70, GST and enolase. In addition, a number of schistosome-specific proteins with unknown function and no homology to any known annotated protein were found to be incorporated in the eggshell. Schistosome-specific glycoconjugates were also shown to be present on the eggshell protein skeleton. This study also confirmed that the putative eggshell protein p14 contributes largely to the eggshell. Together, these results give new insights into eggshell composition as well as eggshell formation. Those proteins that are present at the site and time of eggshell formation are incorporated in the cross-linked eggshell and this cross-linking does no longer occur when the miracidium starts secreting proteins.
During the complex lifecycle of Schistosoma mansoni, a large variety of glycans is expressed. To many of these glycans, antibodies are induced by the infected host and some might be targets for vaccines or diagnostic tests. Spatial changes in glycan expression during schistosome development are largely unexplored. To study the surface-exposed glycans during the important initial stages of infection, we analyzed the binding of a panel of anti-glycan monoclonal antibodies (mAbs) to cercariae and schistosomula up to 72 h after transformation by immunofluorescence microscopy. The mAb specificity toward their natural targets was studied using a microarray containing a wide range of schistosomal N-glycans, O-glycans and glycosphingolipid glycans. With the exception of GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), mono- and multifucosylated GalNAcβ1-4GlcNAc (LDN)-motifs were exposed at the surface of all developmental stages studied. Multifucosylated LDN-motifs were present on cercarial glycocalyx-derived O-glycans as well as cercarial glycolipids. In contrast, the Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and LDN-F-motifs, also expressed on cercarial glycolipids, and in addition on a range of cercarial N- and O-glycans, became surface expressed only after transformation of cercariae to schistosomula. In line with the documented shedding of the O-glycan-rich cercarial glycocalyx after transformation these observations suggest that surface accessible multifucosylated LDN-motifs are mostly expressed by O-glycans in cercariae, but principally by glycosphingolipids in schistosomula. We hypothesize that these temporal changes in surface exposure of glycan antigens are relevant to the interaction with the host during the initial stages of infection with schistosomes and discuss the potential of these glycan antigens as intervention targets.
c Schistosomiasis is a tropical disease affecting over 230 million people worldwide. Although effective drug treatment is available, reinfections are common, and development of immunity is slow. Most antibodies raised during schistosome infection are directed against glycans, some of which are thought to be protective. Developing schistosomula are considered most vulnerable to immune attack, and better understanding of local antibody responses raised against glycans expressed by this life stage might reveal possible glycan vaccine candidates for future vaccine research. We used antibody-secreting cell (ASC) probes to characterize local antiglycan antibody responses against migrating Schistosoma japonicum schistosomula in different tissues of rats. Analysis by shotgun Schistosoma glycan microarray resulted in the identification of antiglycan antibody response patterns that reflected the migratory pathway of schistosomula. Antibodies raised by skin lymph node (LN) ASC probes mainly targeted Nglycans with terminal mannose residues, Gal1-4GlcNAc (LacNAc) and Gal1-4(Fuc␣1-3)GlcNAc (LeX). Also, responses to antigenic and schistosome-specific glycosphingolipid (GSL) glycans containing highly fucosylated GalNAc1-4(GlcNAc1) n stretches that are believed to be present at the parasite's surface constitutively upon transformation were found. Antibody targets recognized by lung LN ASC probes were mainly N-glycans presenting GalNAc1-4GlcNAc (LDN) and GlcNAc motifs. Surprisingly, antibodies against highly antigenic multifucosylated motifs of GSL glycans were not observed in lung LN ASC probes, indicating that these antigens are not expressed in lung stage schistosomula or are not appropriately exposed to induce immune responses locally. The local antiglycan responses observed in this study highlight the stage-and tissue-specific expression of antigenic parasite glycans and provide insights into glycan targets possibly involved in resistance to S. japonicum infection. Schistosomiasis is one of the neglected tropical diseases with the highest impacts on human health. Over 230 million people are infected worldwide, and over 500 million are at risk of infection (1-3). Infection leads to chronic disease characterized by pronounced immunological reactions against eggs deposited into host tissues by the adult schistosome worms, which eventually lead to fibrosis and organ failure (2). Although effective treatment is available, reinfection occurs rapidly and immunity develops only slowly, stressing the need for a prophylactic vaccine as part of a sustainable control strategy (4-6).Schistosomes have a complex life cycle with different life stages that interact with the human host and that each play a role in immunology, immunopathology, and maintenance of infection. Schistosoma infection occurs after direct contact with water containing the larval form of the parasite (cercariae). Cercariae penetrate the host skin and transform into schistosomula, which enter the vasculature and mature while migrating via the lungs to the portal ve...
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