Development and Evaluation of a Microarray Platform for Detection of Serum Antibodies Against Streptococcus pneumoniae Capsular Polysaccharides.

Streptococcus pneumoniae is responsible for severe infections, causing millions of deaths yearly. Immunoglobulin G (IgG) antibodies against the capsular polysaccharide (CPS) offer S. pneumoniae serotype-specific protection. In this work, we examined the applicability of the microarray technology to detect CPS type-specific IgGs in serum, using a collection of 22 microarray-printed S. pneumoniae CPSs. First, printing of five CPSs onto nitrocellulose-coated glass slides was tested. Successful printing was only achieved for certain CPS types and concentrations. This behavior was tentatively related with diverse viscosities of the CPS solutions. Measurement of dynamic viscosities fully supported this assumption and helped to establish suitable CPS type- and concentration-dependent printing conditions. Next, the potential of CPS microarrays for detecting recognition by anti-CPS IgGs was examined using well-defined rabbit pneumococcal antisera. In all cases, the expected antiserum-CPS binding signals were detected, prompting a proof-of-concept analysis of human serum samples. Clearly distinct serum- and CPS-specific binding patterns and intensities were observed, evidencing selective detection of CPS type-specific IgGs. Compared to the ELISA assay commonly used to quantitate CPS type-specific IgGs in serum, the newly developed S. pneumoniae CPS microarrays offer the advantage of enabling the simultaneous analysis of multiple CPS-serum interactions using minute CPS amounts and significantly reduced serum volumes. Therefore, the approach could be particularly valuable for gauging the presence of CPS type-specific IgGs in human serum when sample volumes are limited and/or numerous serum samples are being examined.

Combined Bacteria Microarray and Quartz Crystal Microbalance Approach for Exploring Glycosignatures of Nontypeable Haemophilus influenzae and Recognition by Host Lectins.

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.

The C-type lectin receptor CLECSF8 (CLEC4D) is expressed by myeloid cells and triggers cellular activation through Syk kinase.

CLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor.

The role of sialyl glycan recognition in host tissue tropism of the avian parasite Eimeria tenella.

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates.

Early murine T-lymphocyte activation is accompanied by a switch from N-Glycolyl- to N-acetyl-neuraminic acid and generation of ligands for siglec-E.

It is well established that murine T-lymphocyte activation is accompanied by major changes in cell-surface sialylation, potentially influencing interactions with sialic acid-binding immunoglobulin-like lectins (siglecs). In the present study, we analyzed early activation of murine CD4+ and CD8+ T-lymphocytes at 24 h. We observed a striking and selective up-regulation in the binding of a recombinant soluble form of siglec-E, an inhibitory siglec, which is expressed on several myeloid cell types including antigen-presenting dendritic cells. In contrast, much lower levels of T cell binding were observed with other siglecs, including sialoadhesin, CD22, and siglec-F and the plant lectins Maackia amurensis leukoagglutinin and Sambucus nigra agglutinin. By mass spectrometry, the sialic acid content of 24-h-activated CD4+ and CD8+ T-lymphocytes exhibited an increased proportion of N-acetyl-neuraminic acid (NeuAc) to N-glycolyl-neuraminic acid (NeuGc) in N-glycans. Reduced levels of NeuGc on the surface of activated T cells were demonstrated using an antibody specific for NeuGc and the expression levels of the gene encoding NeuAc- to NeuGc-converting enzyme, CMP-NeuAc hydroxylase, were also reduced. Siglec-E bound a wide range of sialylated structures in glycan arrays, had a preference for NeuAc versus NeuGc-terminated sequences and could recognize a set of sialoglycoproteins that included CD45, in lysates from activated T-lymphocytes. Collectively, these results show that early in T cell activation, glycan remodelling involves a switch from NeuGc- to NeuAc-terminating oligosaccharides on cell surface glycoproteins. This is associated with a strong up-regulation of siglec-E ligands, which may be important in promoting cellular interactions between early activated T-lymphocytes and myeloid cells expressing this inhibitory receptor.

An expression system for screening of proteins for glycan and protein interactions.

Here we describe a versatile high-throughput expression system that permits genome-wide screening of type 1 membrane and secreted proteins for interactions with glycans and proteins using both cell-expressed and soluble forms of the expressed proteins. Based on Gateway cloning methodology, we have engineered a destination vector that directs expression of enhanced green fluorescent protein (EGFP)-tagged proteins at the cell surface via a glycosylphosphatidylinositol tail. The EGFP fusion proteins can then be cleaved with PreScission protease to release soluble forms of proteins that can be optionally biotinylated. We demonstrate the utility of this cloning and expression system for selected low-affinity membrane lectins from the siglec family of sialic acid-binding immunoglobulin-like lectins, for the glycosaminoglycan-binding proteins FGF-1 and BACE, and for the heterotypic adhesion molecules JAM-B and JAM-C. Cell-expressed proteins can be evaluated for glycan interactions using polyvalent soluble glycan probes and for protein interactions using either cells or soluble proteins. Following cleavage from the cell surface, proteins were complexed in solution and sufficient avidity was achieved to measure weak protein-glycan and weak protein-protein interactions using glycan arrays and surface plasmon resonance, respectively.

Exploration of Galectin Ligands Displayed on Gram-Negative Respiratory Bacterial Pathogens with Different Cell Surface Architectures.

Galectins bind various pathogens through recognition of distinct carbohydrate structures. In this work, we examined the binding of four human galectins to the Gram-negative bacteria Klebsiella pneumoniae (Kpn) and non-typeable Haemophilus influenzae (NTHi), which display different surface glycans. In particular, Kpn cells are covered by a polysaccharide capsule and display an O-chain-containing lipopolysaccharide (LPS), whereas NTHi is not capsulated and its LPS, termed lipooligosacccharide (LOS), does not contain O-chain. Binding assays to microarray-printed bacteria revealed that galectins-3, -4, and -8, but not galectin-1, bind to Kpn and NTHi cells, and confocal microscopy attested binding to bacterial cells in suspension. The three galectins bound to array-printed Kpn LPS. Moreover, analysis of galectin binding to mutant Kpn cells evidenced that the O-chain is the docking point for galectins on wild type Kpn. Galectins-3, -4, and -8 also bound the NTHi LOS. Microarray-assisted comparison of the binding to full-length and truncated LOSs, as well as to wild type and mutant cells, supported LOS involvement in galectin binding to NTHi. However, deletion of the entire LOS oligosaccharide chain actually increased binding to NTHi cells, indicating the availability of other ligands on the bacterial surface, as similarly inferred for Kpn cells devoid of both O-chain and capsule. Altogether, the results illustrate galectins' versatility for recognizing different bacterial structures, and point out the occurrence of so far overlooked galectin ligands on bacterial surfaces.

N-glycolyl GM1 ganglioside as a receptor for simian virus 40.

Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions.

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewis(x), (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

The Role of Collectins and Galectins in Lung Innate Immune Defense.

Different families of endogenous lectins use complementary defense strategies against pathogens. They may recognize non-self glycans typically found on pathogens and/or host glycans. The collectin and galectin families are prominent examples of these two lectin categories. Collectins are C-type lectins that contain a carbohydrate recognition domain and a collagen-like domain. Members of this group include surfactant protein A (SP-A) and D (SP-D), secreted by the alveolar epithelium to the alveolar fluid. Lung collectins bind to several microorganisms, which results in pathogen aggregation and/or killing, and enhances phagocytosis of pathogens by alveolar macrophages. Moreover, SP-A and SP-D influence macrophage responses, contributing to resolution of inflammation, and SP-A is essential for tissue-repair functions of macrophages. Galectins also function by interacting directly with pathogens or by modulating the immune system in response to the infection. Direct binding may result in enhanced or impaired infection of target cells, or can have microbicidal effects. Immunomodulatory effects of galectins include recruitment of immune cells to the site of infection, promotion of neutrophil function, and stimulation of the bactericidal activity of infected macrophages. Moreover, intracellular galectins can serve as danger receptors, promoting autophagy of the invading pathogen. This review will focus on the role of collectins and galectins in pathogen clearance and immune response activation in infectious diseases of the respiratory system.

Bacterial Surface Glycans: Microarray and QCM Strategies for Glycophenotyping and Exploration of Recognition by Host Receptors.

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment.

Deciphering how Cpl-7 cell wall-binding repeats recognize the bacterial peptidoglycan.

Endolysins, the cell wall lytic enzymes encoded by bacteriophages to release the phage progeny, are among the top alternatives to fight against multiresistant pathogenic bacteria; one of the current biggest challenges to global health. Their narrow range of susceptible bacteria relies, primarily, on targeting specific cell-wall receptors through specialized modules. The cell wall-binding domain of Cpl-7 endolysin, made of three CW_7 repeats, accounts for its extended-range of substrates. Using as model system the cell wall-binding domain of Cpl-7, here we describe the molecular basis for the bacterial cell wall recognition by the CW_7 motif, which is widely represented in sequences of cell wall hydrolases. We report the crystal and solution structure of the full-length domain, identify N-acetyl-D-glucosaminyl-(ß1,4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) as the peptidoglycan (PG) target recognized by the CW_7 motifs, and characterize feasible GMDP-CW_7 contacts. Our data suggest that Cpl-7 cell wall-binding domain might simultaneously bind to three PG chains, and also highlight the potential use of CW_7-containing lysins as novel anti-infectives.

Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses.

Carbohydrate microarrays, since their advent in 2002, are revolutionizing studies of the molecular basis of protein-carbohydrate interactions both in endogenous recognition systems and pathogen-host interactions. We have developed a unique carbohydrate microarray system based on the neoglycolipid (NGL) technology, a well-validated microscale approach for generating lipid-tagged oligosaccharide probes for use in carbohydrate recognition studies. This chapter provides an overview of the principles and key features of the NGL-based oligosaccharide microarrays, and describes in detail the basic techniques - from the preparation of NGL probes to the generation of microarrays using robotic arraying hardware, as well as a general protocol for probing the microarrays with carbohydrate-binding proteins.

Neoglycolipid-based "designer" oligosaccharide microarrays to define ß-glucan ligands for Dectin-1.

In this chapter, we describe the key steps of the "designer" oligosaccharide microarray approach we followed to prove the carbohydrate binding activity and define the oligosaccharide ligands for Dectin-1, an atypical C-type lectin-like signaling receptor of the mammalian innate immune system with a key role in anti-fungal immunity. The term "designer" microarray, which we introduced in the course of the Dectin-1 study refers to a microarray of oligosaccharide probes generated from ligand-bearing glycoconjugates to reveal the oligosaccharide ligands they harbor, so that these can be isolated and characterized. Oligosaccharide probes were generated from two polysaccharides, one that was bound by Dectin-1 and known to be rich in ß1,3-glucose sequence and another that was not bound and was rich in ß1,6-glucose sequence and served as a negative control. The approach involved: classic ELISA-type binding assays to select the polysaccharides; partial depolymerization of the polysaccharides by chemical hydrolysis; fractionation by size of the glucan oligosaccharides obtained and determination of their chain lengths by mass spectrometry; detection of Dectin-1 ligand-positive and ligand-negative oligosaccharides using the neoglycolipid (NGL) technology; methylation analysis of oligosaccharides to derive glucose linkage information, and incorporation of the newly generated glucan oligosaccharide probes into microarrays encompassing diverse mammalian-type and exogenous sequences for microarray analysis of Dectin-1.

Atomic resolution insight into host cell recognition by Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell-binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high-resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein. We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite-receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies.

Zinc ions induce the unfolding and self-association of boar spermadhesin PSP-I, a protein with a single CUB domain architecture, and promote its binding to heparin.

Spermadhesins are a family of seminal plasma proteins composed of a single CUB domain, which appear to be involved in various aspects of the fertilization process in pigs. PSP-I and PSP-II, the most abundant porcine spermadhesins, occur in seminal plasma as noncovalent heterodimers devoid of heparin-binding capability. Of note is the stability of this dimer, which is significantly affected by physiologically relevant conditions such as Zn2+ ions. Here, we show that PSP-I and PSP-II when separated appear to conserve the overall fold of the CUB domain observed in the crystal structure of the PSP-I/PSP-II heterodimer, as concluded from gel filtration, analytical ultracentrifugation, differential scanning calorimetry, and circular dichroism analyses. However, Zn2+ concentrations in the range of those found in boar seminal plasma induce the unfolding and self-association of PSP-I, apparently as a consequence of the exposure of hydrophobic core residues, whereas they have no effect on PSP-II. Remarkably, Zn2+-denatured and self-associated (but not structured monomeric) PSP-I is retained on a heparin column, resembling the behavior of free PSP-I and homologous spermadhesins of the heparin-binding fraction of boar seminal plasma, which also exhibit different aggregation states. Thus, the modulation of the structural organization and heparin-binding ability of PSP-I by Zn2+ might be a physiological phenomenon in seminal plasma.