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.

Direct Evaluation of Live Uropathogenic Escherichia coli Adhesion and Efficiency of Antiadhesive Compounds Using a Simple Microarray Approach.

Many pathogens use host glycans as docking points for adhesion. Therefore, the use of compounds blocking carbohydrate-binding adhesins is a promising strategy for fighting infections. In this work, we describe a simple and rapid microarray approach for assessing the bacterial adhesion and efficiency of antiadhesive compounds targeting uropathogenic Escherichia coli UTI89, which displays mannose-specific adhesin FimH at the tip of fimbriae. The approach consisted in direct detection of live fluorescently labeled bacteria bound to mannan printed onto microarray slides. The utility of the arrays for binding/inhibition assays was first validated by comparing array-derived results for the model mannose-binding lectin concanavalin A with data obtained by isothermal titration calorimetry. Growth phase-dependent binding of UTI89 to the arrays was observed, proving the usefulness of the setup for detecting differences in FimH expression. Importantly, bacteria labeling and binding assays entailed minimal manipulation, helping to preserve the integrity of fimbriae. The efficiency of three different dodecamannosylated fullerenes as FimH-targeted antiadhesives was next evaluated in competition assays. The results revealed a superior activity of the mannofullerenes (5- to 18-fold per mannose residue) over methyl α-d-mannopyranoside. Moreover, differences in activity were detected for mannofullerenes differing in the structure/length of the spacer used for grafting mannose onto the fullerene core, further demonstrating the sensitivity of the assay. Overall, the approach combines straightforward and time-saving protocols for microarray preparation, bacteria labeling, and binding assays, and it can be easily tailored to other bacteria bearing carbohydrate-binding adhesins.

A guide into glycosciences: How chemistry, biochemistry and biology cooperate to crack the sugar code.

BACKGROUND: The most demanding challenge in research on molecular aspects within the flow of biological information is posed by the complex carbohydrates (glycan part of cellular glycoconjugates). How the 'message' encoded in carbohydrate 'letters' is 'read' and 'translated' can only be unraveled by interdisciplinary efforts. SCOPE OF REVIEW: This review provides a didactic step-by-step survey of the concept of the sugar code and the way strategic combination of experimental approaches characterizes structure-function relationships, with resources for teaching. MAJOR CONCLUSIONS: The unsurpassed coding capacity of glycans is an ideal platform for generating a broad range of molecular 'messages'. Structural and functional analyses of complex carbohydrates have been made possible by advances in chemical synthesis, rendering production of oligosaccharides, glycoclusters and neoglycoconjugates possible. This availability facilitates to test the glycans as ligands for natural sugar receptors (lectins). Their interaction is a means to turn sugar-encoded information into cellular effects. Glycan/lectin structures and their spatial modes of presentation underlie the exquisite specificity of the endogenous lectins in counterreceptor selection, that is, to home in on certain cellular glycoproteins or glycolipids. GENERAL SIGNIFICANCE: Understanding how sugar-encoded 'messages' are 'read' and 'translated' by lectins provides insights into fundamental mechanisms of life, with potential for medical applications.

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.

From lectin structure to functional glycomics: principles of the sugar code.

Lectins are carbohydrate-binding proteins which lack enzymatic activity on their ligand and are distinct from antibodies and free mono- and oligosaccharide sensor/transport proteins. Emerging insights into the functional dimension of lectin binding to cellular glycans have strongly contributed to the shaping of the 'sugar code'. Fittingly, over a dozen folds and a broad spectrum of binding site architecture, ranging from shallow grooves to deep pockets, have developed sugar-binding capacity. A central question is how the exquisite target specificity of endogenous lectins for certain cellular glycans can be explained. In this regard, affinity regulation is first systematically dissected into six levels. Experimentally, the strategic combination of methods to monitor distinct aspects of the lectin-glycan interplay offers a promising perspective to answer this question.

Insights into the recognition of hypermucoviscous Klebsiella pneumoniae clinical isolates by innate immune lectins of the Siglec and galectin families.

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

Structure and properties of the exopolysaccharide isolated from Flavobacterium sp. Root935.

Flavobacterium strains exert a substantial influence on roots and leaves of plants. However, there is still limited understanding of how the specific interactions between Flavobacterium and their plant hosts are and how these bacteria thrive in this competitive environment. A crucial step in understanding Flavobacterium - plant interactions is to unravel the structure of bacterial envelope components and the molecular features that facilitate initial contact with the host environment. Here, we have revealed structure and properties of the exopolysaccharides (EPS) produced by Flavobacterium sp. Root935. Chemical analyses revealed a complex and interesting branched heptasaccharidic repeating unit, containing a variety of sugar moieties, including Rha, Fuc, GlcN, Fuc4N, Gal, Man and QuiN and an important and extended substitution pattern, including acetyl and lactyl groups. Additionally, conformational analysis using molecular dynamics simulation showed an extended hydrophobic interface and a distinctly elongated, left-handed helicoidal arrangement. Furthermore, properties of the saccharide chain, and likely the huge substitution pattern prevented interaction and recognition by host lectins and possessed a low immunogenic potential, highlighting a potential role of Flavobacterium sp. Root935 in plant-microbial crosstalk.

Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity.

The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of (15)N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 10(3) M(-1) and K2 = 3.4 ± 0.8 × 10(3) M(-1). Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.

Fine-tuning of prototype chicken galectins: structure of CG-2 and structure-activity correlations.

The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study the divergence of these lectins after gene duplication. Expression profiling of the three homodimeric (prototype) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence of distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identify differential changes in the circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behaviour, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism.

Atomic crystal structure and sugar specificity of a ß-trefoil lectin domain from the ectomycorrhizal basidiomycete Laccaria bicolor.

Lectins from fruiting bodies are a diverse group of sugar-binding proteins from mushrooms that face the biologically relevant challenge of discriminating self- from non-self carbohydrate structures, therefore providing a basis for an innate defence system. Such a system entails both detection and destruction of invaders and/or feeders, and in contrast to more complex organisms with immense immune systems, these two functions normally rely on multitasking lectins, namely, lectins with different functional modules. Here, we present a novel fungal lectin, LBL, from the basidiomycete Laccaria bicolor. Using a diverse set of biophysical techniques, we unveil the fine details of the sugar-binding specificity of the N-terminal ß-trefoil of LBL (LBL152), whose structure has been determined at the highest resolution so far reported for such a fold. LBL152 binds complex poly-N-Acetyllactosamine polysaccharides and also robust LBL152 binding to Caenorhabditis elegans and Drosophila melanogaster cellular extracts was detected in microarray assays, with a seeming preference for the fruit fly adult and pupa stages over the larva stage. Prediction of the structure of the C-terminal part of LBL with AlphaFold reveals a tandem repeat of two structurally almost identical domains of around 110 amino acids each, despite sharing low sequence conservation.

Bacterial Microarrays for Examining Bacterial Glycosignatures and Recognition by Host Lectins.

The surface of bacteria displays diverse carbohydrate structures that may significantly differ among bacteria with the same cell wall architecture and even among strains of a given bacterial species. These structures are often recognized by lectins of the innate immune system for triggering defense responses, although some bacterial pathogens exploit recognition by host lectins for favoring infection. Bacterial microarrays are a useful tool for profiling accessible bacterial surface glycans and for exploring their recognition by innate immune lectins. The use of array-printed bacterial cells enables evaluation of the recognition of the glycan epitopes in their natural presentation, i.e., preserving their real density and accessibility. Glycosylation patterns of bacterial surfaces can be examined by testing the binding to the bacterial arrays of a panel of lectins with known carbohydrate-binding preferences, and the recognition of surface glycans by innate immune lectins can easily be assessed using similar binding assays.

Lipopolysaccharide O-antigen molecular and supramolecular modifications of plant root microbiota are pivotal for host recognition.

Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated D-rhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O-deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition.

Symmetric dithiodigalactoside: strategic combination of binding studies and detection of selectivity between a plant toxin and human lectins.

Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (K(a): 1560 ± 20 M(-1)). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested glycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design.

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.

Unique chicken tandem-repeat-type galectin: implications of alternative splicing and a distinct expression profile compared to those of the three proto-type proteins.

Animal galectins (lectins with specificity for beta-galactosides of glycan chains) are potent effectors in diverse aspects of cell sociology. Gene divergence has led to different groups and a marked interspecies variability in the number of members per group. Since the suitability of a model for studying functionality in the galectin network will be distinguished by a rather simple degree of complexity, we have focused on chicken galectins (CGs). Starting from partial expression sequence tag information, we here report on cloning of full-length cDNA for the first avian tandem-repeat-type galectin. It is termed CG-8 on the basis of its sequence similarity to galectin-8 from mammals. Systematic sequence searches revealed its unique character among CGs. Detection of two mature mRNA species points to production of isoforms. Alternative splicing affecting exon V generates the two proteins with linkers of either 9 (CG-8I) or 28 amino acids (CG-8II). Both proteins form monomers with a shape comparable to that of the proto-type proteins CG-1A/B in solution, act as cross-linkers in hemagglutination, and bind cells with a strict dependence on galactose. Western blotting revealed the presence of either CG-8II or the mixture in organ extracts. No evidence of a truncated form was obtained. Preparation of a specific antibody also enabled immunohistochemical localization. Prominent sites of its presence were defense cells in the l. propria mucosae, in addition to immune cells in distinct organs such as alveolar macrophages and thymocytes. Overall, we extend the network of CGs to a tandem-repeat-type protein and provide a detailed characterization from gene and protein structures to expression.

Prototype chicken galectins revisited: characterization of a third protein with distinctive hydrodynamic behaviour and expression pattern in organs of adult animals.

Prototype galectins are versatile modulators of cell adhesion and growth via their reactivity to certain carbohydrate and protein ligands. These functions and the galectins' marked developmental regulation explain their attractiveness as models to dissect divergent evolution after gene duplication. Only two members have so far been assumed to constitute this group in chicken, namely the embryonic muscle/liver form {C-16 or CLL-I [16 kDa; chicken lactose lectin, later named CG-16 (chicken galectin-16)]} and the embryonic skin/intestine form (CLL-II or C-14; later named CG-14). In the present study, we report on the cloning and expression of a third prototype CG. It has deceptively similar electrophoretic mobility compared with recombinant C-14, the protein first isolated from embryonic skin, and turned out to be identical with the intestinal protein. Hydrodynamic properties unusual for a homodimeric galectin and characteristic traits in the proximal promoter region set it apart from the two already known CGs. Their structural vicinity to galectin-1 prompts their classification as CG-1A (CG-16)/CG-1B (CG-14), whereas sequence similarity to mammalian galectin-2 gives reason to refer to the intestinal protein as CG-2. The expression profiling by immunohistochemistry with specific antibodies discerned non-overlapping expression patterns for the three CGs in several organs of adult animals. Overall, the results reveal a network of three prototype galectins in chicken.

Microarray Strategies for Exploring Bacterial Surface Glycans and Their Interactions With Glycan-Binding Proteins.

Bacterial surfaces are decorated with distinct carbohydrate structures that may substantially differ among species and strains. These structures can be recognized by a variety of glycan-binding proteins, playing an important role in the bacteria cross-talk with the host and invading bacteriophages, and also in the formation of bacterial microcolonies and biofilms. In recent years, different microarray approaches for exploring bacterial surface glycans and their recognition by proteins have been developed. A main advantage of the microarray format is the inherent miniaturization of the method, which allows sensitive and high-throughput analyses with very small amounts of sample. Antibody and lectin microarrays have been used for examining bacterial glycosignatures, enabling bacteria identification and differentiation among strains. In addition, microarrays incorporating bacterial carbohydrate structures have served to evaluate their recognition by diverse host/phage/bacterial glycan-binding proteins, such as lectins, effectors of the immune system, or bacterial and phagic cell wall lysins, and to identify antigenic determinants for vaccine development. The list of samples printed in the arrays includes polysaccharides, lipopoly/lipooligosaccharides, (lipo)teichoic acids, and peptidoglycans, as well as sequence-defined oligosaccharide fragments. Moreover, microarrays of cell wall fragments and entire bacterial cells have been developed, which also allow to study bacterial glycosylation patterns. In this review, examples of the different microarray platforms and applications are presented with a view to give the current state-of-the-art and future prospects in this field.

Domain versatility in plant AB-toxins: evidence for a local, pH-dependent rearrangement in the 2gamma lectin site of the mistletoe lectin by applying ligand derivatives and modelling.

Mistletoe lectin is a potent biohazard. Lectin activity in the toxic dimer primarily originates from the 2gamma-subdomain (Tyr-site) of the B-subunit. Crystallographic information on lectin-sugar complexes is available only at acidic pH, where lectin activity is low. Thus, we mapped ligand-binding properties including comparison to ricin's Tyr-site at neutral pH. Using these results and molecular dynamics simulations, a local conformational change was rendered likely. The obtained structural information is valuable for the design of potent inhibitors.

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.