Angela van Diepen

Differential anti-glycan antibody responses in Schistosoma mansoni-infected children and adults studied by shotgun glycan microarray.

Background Schistosomiasis (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 Findings The 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/Significance Using 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.

Glycomic analysis of life stages of the human parasite Schistosoma mansoni reveals developmental expression profiles of functional and antigenic glycan motifs.

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 schistosomes glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.

Complement receptor 3 and toll-like receptor 4 act sequentially in uptake and intracellular killing of unopsonized Salmonella enterica serovar Typhimurium by human neutrophils

ABSTRACT The uptake and subsequent killing of Salmonella enterica serovar Typhimurium by human neutrophils was studied. In particular, two pattern recognition receptors, complement receptor 3 (CR3) and Toll-like receptor 4 (TLR4), were found to be essential for the efficient uptake and activation, respectively, of the NADPH oxidase. The uptake of Salmonella was almost completely inhibited by various monoclonal antibodies against CR3, and neutrophils from a patient with leukocyte adhesion deficiency type 1, which lack CR3, showed almost no uptake of Salmonella. A lipopolysaccharide (LPS) mutant strain of Salmonella was used to show that the expression of full-length, wild-type, or so-called smooth LPS is important for the efficient killing of intracellular Salmonella. Infection with wild-type-LPS-expressing Salmonella resulted in the generation of reactive oxygen species (ROS) in TLR4-decorated, Salmonella-containing vacuoles, whereas ROS were not induced by an LPS mutant strain. In addition, the recognition of Salmonella by neutrophils, leading to ROS production, was shown to be intracellular, as determined by priming experiments with intact bacteria under conditions where the bacterium is not taken up. Finally, the generation of ROS in the wild-type-Salmonella-infected neutrophils was largely inhibited by the action of a TLR4-blocking, cell-permeable peptide, showing that signaling by this receptor from the Salmonella-containing vacuole is essential for the activation of the NADPH oxidase. In sum, our data identify the sequential recognition of unopsonized Salmonella strains by CR3 and TLR4 as essential events in the efficient uptake and killing of this intracellular pathogen.

Parasite glycans and antibody-mediated immune responses in Schistosoma infection

Schistosome infections in humans are characterized by the development of chronic disease and high re-infection rates after treatment due to the slow development of immunity. It appears that anti-schistosome antibodies are at least partially mediating protective mechanisms. Efforts to develop a vaccine based on immunization with surface-exposed or secreted larval or worm proteins are ongoing. Schistosomes also express a large number of glycans as part of their glycoprotein and glycolipid repertoire, and antibody responses to those glycans are mounted by the infected host. This observation raises the question if glycans might also form novel vaccine targets for immune intervention in schistosomiasis. This review summarizes current knowledge of antibody responses and immunity in experimental and natural infections with Schistosoma, the expression profiles of schistosome glycans (the glycome), and antibody responses to individual antigenic glycan motifs. Future directions to study anti-glycan responses in schistosomiasis in more detail in order to address more precisely the possible role of glycans in antibody-mediated immunity are discussed.

A Superoxide-Hypersusceptible Salmonella enterica Serovar Typhimurium Mutant Is Attenuated but Regains Virulence in p47phox−/− Mice

ABSTRACT Salmonella enterica serovar Typhimurium is a gram-negative, facultative intracellular pathogen that predominantly invades mononuclear phagocytes and is able to establish persistent infections. One of the innate defense mechanisms of phagocytic cells is the production of reactive oxygen species, including superoxide. S. enterica serovar Typhimurium has evolved mechanisms to resist such radicals, and these mechanisms could be decisive in its ability to survive and replicate within macrophages. Recently, we described a superoxide-hypersusceptible S. enterica serovar Typhimurium mutant strain, DLG294, that carries a transposon in sspJ, resulting in the lack of expression of SspJ, which is necessary for resistance against superoxide and replication within macrophages. Here we show that DLG294, which is a 14028s derivative, hardly induced any granulomatous lesions in the livers upon subcutaneous infection of C3H/HeN (Ityr) mice with 3 × 104 bacteria and that its bacterial counts were reduced by 3 log units compared to those of wild-type S. enterica serovar Typhimurium 14028s on day 5 after infection. In contrast, DLG294 replicated like wild-type S. enterica serovar Typhimurium 14028s and induced a phenotypically similar liver pathology in p47phox−/− mice, which are deficient in the p47phox subunit of the NADPH oxidase complex and which do not produce superoxide. Consistent with these results, DLG294 reached bacterial counts identical to those of wild-type S. enterica serovar Typhimurium 14028s in bone marrow-derived macrophages from p47phox−/− mice and in X-CGD PLB-985 cells at 24 h after challenge. These results indicate that SspJ plays a role in the bacteriums resistance to oxidative stress and in the survival and replication of S. enterica serovar Typhimurium both in vitro and in vivo.

Transcriptome kinetics of circulating neutrophils during human experimental endotoxemia.

Polymorphonuclear cells (neutrophils) play an important role in the systemic inflammatory response syndrome and the development of sepsis. These cells are essential for the defense against microorganisms, but may also cause tissue damage. Therefore, neutrophil numbers and activity are considered to be tightly regulated. Previous studies have investigated gene transcription during experimental endotoxemia in whole blood and peripheral blood mononuclear cells. However, the gene transcription response of the circulating pool of neutrophils to systemic inflammatory stimulation in vivo is currently unclear. We examined neutrophil gene transcription kinetics in healthy human subjects (n = 4) administered a single dose of endotoxin (LPS, 2 ng/kg iv). In addition, freshly isolated neutrophils were stimulated ex vivo with LPS, TNFα, G-CSF and GM-CSF to identify stimulus-specific gene transcription responses. Whole transcriptome microarray analysis of circulating neutrophils at 2, 4 and 6 hours after LPS infusion revealed activation of inflammatory networks which are involved in signaling of TNFα and IL-1α and IL-1β. The transcriptome profile of inflammatory activated neutrophils in vivo reflects extended survival and regulation of inflammatory responses. These changes in neutrophil transcriptome suggest a combination of early activation of circulating neutrophils by TNFα and G-CSF and a mobilization of young neutrophils from the bone marrow.

Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

Development of a Schistosoma mansoni shotgun O-glycan microarray and application to the discovery of new antigenic schistosome glycan motifs

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets.

Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

Gamma Irradiation or CD4-T-Cell Depletion Causes Reactivation of Latent Salmonella enterica Serovar Typhimurium Infection in C3H/HeN Mice

ABSTRACT Upon infection with Salmonella, a host develops an immune response to limit bacterial growth and kill and eliminate the pathogen. Salmonella has evolved mechanisms to remain dormant within the body, only to reappear (reactivate) at a later time when the immune system is abated. We have developed an in vivo model for studying reactivation of Salmonella enterica serovar Typhimurium infection in mice. Upon subcutaneous infection, C3H/HeN (Ityr) mice showed an increase in bacterial numbers in livers and spleens, which reached a peak on day 19. After full recovery from the infection, these mice were irradiated or depleted of CD4+ T cells. The mice displayed a secondary infection peak in livers and spleens with a course similar to that of the primary infection. We concluded that CD4+ T cells are involved in active suppression of S. enterica serovar Typhimurium during latency. The role of CD4+ T cells during primary infection with S. enterica serovar Typhimurium is well established. This is the first study to describe a role of CD4+ T cells during the latent phase of S. enterica serovar Typhimurium infection.