Abstract Neisseria gonorrhoeae causes gonorrhea and poses a growing global health threat driven by antimicrobial resistance and the lack of an effective vaccine. The lipooligosaccharide (LOS) epitope recognized by monoclonal antibody (mAb) 2C7 is expressed by most clinical isolates, making it an attractive therapeutic target. To determine the molecular basis for mAb 2C7 recognition, we conducted structural studies of mAb 2C7 in complex with an octasaccharide derived from LOS. NMR epitope mapping demonstrated that mAb 2C7 contacts two of three glycan chains: the β-chain (lactose) and the γ-chain (N-acetylglucosamine). The 1.6-Å resolution crystal structure of the Fab 2C7–octasaccharide complex revealed that these chains make extensive hydrogen bonds and hydrophobic contacts within a cleft of the Fab. Molecular dynamics (MD) simulations showed that the β and γ chains adopt stable, specific conformations, contrasted with transient interactions of the α chain. Informed by these structural data, we synthesized a tetrasaccharide comprising the β and γ chains joined by heptose II. The tetrasaccharide conjugated to a carrier protein bound mAb 2C7 by ELISA and Western blotting. Isothermal titration calorimetry showed that the tetrasaccharide and octasaccharide bound mAb 2C7 with similar affinities, indicating that the smaller oligosaccharide retains the key binding determinants. Binding was enthalpically driven, consistent with the polar interactions observed crystallographically and by MD. NMR experiments with the tetrasaccharide confirmed interactions with all four residues, establishing it as the minimal 2C7 epitope. Together, these studies provide a structural framework for rational design of glycan-based vaccines and antibody therapeutics against antimicrobial-resistant N. gonorrhoeae .

Ligand design and synthesis for Siglec-8 is important due to its roles in immune responses to allergens, chronic inflammatory disorders and malignancies. Here the synthesis, from readily accessible intermediates, of glycomimetics of Neu5Acα2-3 (6-O-sulfo-Gal), the minimal glycan ligand for Siglec-8, is reported. The mimetics retain Neu5Ac, while replacing the 6-O-sulfo-Gal component by a triazole containing linker presenting either carboxylate or sulfonate groups. Isothermal calorimetry (ITC) measurements showed that the sialyl triazole derivatives were ligands for Siglec-8, with a Kd of 45.5 μM established for the best ligand from the series. The ITC measurements showed that sulfonate was better than carboxylate as a replacement for sulfate, while the incorporation of the naphthylsulfonate group at the Neu5Ac C-9 of the ligands gave affinity improvement, consistent with earlier research. An NMR based binding study supports the conformational change to Siglec-8 to accommodate the 9-naphthylsulfonate group and indicates a similar mode of binding for the highest affinity triazole derivative to previously reported cyclohexane based glycomimetics. The perturbation of NMR signals, docking and binding pose metadynamics support binding of the ligand to the carbohydrate recognition domain. The research provides new Siglec-8 ligands from readily accessible intermediates as a basis for further development.

Bacterial glycoconjugates are central players at the host–microbe interface. Their remarkable structural diversity underlies broad biological functions but also complicates their molecular characterization. This review highlights NMR spectroscopy and computational methods as essential tools to address this complexity. NMR provides atomic-level insights into structural details, conformational dynamics, and binding epitopes. Computational approaches complement these results, predicting conformations, refining interaction models, and linking flexibility to recognition events. Combined with additional biophysical techniques, these strategies enable a multidisciplinary framework for investigating the structure and conformation of bacterial glycoconjugates as well as the molecular basis of their interaction with host receptors. Case studies illustrate how the synergy of experimental and theoretical methods can come to the aid of providing high-resolution models that connect molecular structure with biological functions. Looking ahead, this integrated approach is crucial for disclosing the structural basis of recognition by host proteins, clarifying mechanisms of immune evasion, and defining protective epitopes relevant for vaccine design, thus promising translational applications, including diagnostics, immunotherapy, and the rational design of next-generation antimicrobial strategies.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are key immune receptors that bind to cell surface sialic acids, leading to modulation of the immune system. Interrupting the Siglec-sialoglycan binding in cancer has been proposed as a potential antitumor response strategy. We previously showed that colon-cancer-associated Fusobacterium nucleatum (Fn) ATCC 51191 interacts with Siglec-7 via its lipopolysaccharide (LPS), revealing Fn LPS as a new ligand for Siglec-7. Here, we used glycoengineered cells carrying sialic acid variants to investigate the capacity of LPS isolated from F. nucleatum strains to disrupt the interaction between sialic acid expressed on mammalian cells and Siglec-7. We first showed that LPS extracted from Fusobacterium polymorphum ATCC 10953, F. nucleatum ssp. animalis ATCC 51191, and F. nucleatum ssp. nucleatum ATCC 25586 strains bound to recombinant Siglec-7 in vitro, while no binding was observed with the Siglec-7R124A mutant, suggesting that the binding occurred through the carbohydrate binding V-set domain. Using glycoengineered Jurkat cells and HEK293T cells carrying modified sialic acid forms, we demonstrated that F. nucleatum LPS could significantly disrupt the binding of Siglec-7 to these cells and that this inhibition was decreased following neuraminidase treatment, confirming that the interaction between Fn LPS and Siglec-7 is carbohydrate-mediated. We further validated these data using Jurkat cells and HEK293T cells expressing high-affinity sialic acid ligands. We showed that F. nucleatum LPS significantly disrupted the binding of Siglec-7 to these cells in a specific manner. These findings offer novel insights into the development of glycomimetic approaches for limiting colon cancer progression.

Deciphering the role(s) of surface glycoconjugates in gut microbiota-immune interactions is essential for improving human health. This study shows that Bacteroides stercoris DSM 19555 produces two previously undescribed surface glycoconjugates: an atypical rough-type lipopolysaccharide (R-LPS) and a positively charged capsular polysaccharide (CPS). Structural analysis revealed an R-LPS featuring a 2-aminoethyl phosphate-substituted 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo) bearing a phosphodiester-linked hexose, a fucose-lacking core oligosaccharide, and a highly heterogeneous mono-phosphorylated, hypoacylated lipid A. In parallel, a branched, galacto-configured, and amino sugar-rich cationic CPS is also identified. Both glycoconjugates promote anti-inflammatory IL-10 production while limiting classical pro-inflammatory outputs, with CPS exhibiting the strongest immunomodulatory activity. In an inflamed epithelial-immune coculture model, both glycoconjugates shape cytokine responses toward an IL-10-associated, low-inflammatory profile. These findings uncover a structurally and functionally distinct glycan repertoire in B. stercoris DSM 19555 and support the emerging view that gut Bacteroidales surface glycans behave as active modulators of innate immune responses rather than as simple inflammatory triggers.

Lipopolysaccharides (LPSs) from Gram-negative bacteria are widely used to model neuroinflammation in vitro and in vivo. However, this paradigm assumes that all LPS chemotypes are uniformly pro-inflammatory, despite significant structural diversity between enterobacterial pathogens and gut-resident commensals. Whether microglia can discriminate among these chemotypes remains largely unexplored. We performed a comparative analysis of canonical Escherichia coli LPS and commensal-derived Phocaeicola vulgatus LPS in murine (BV2) and human (HMC3) microglial cells. Pro-inflammatory mediators were quantified by ELISA, and TLR4-downstream signaling was assessed by western blotting. Conditioned media (CM) from LPS-treated BV2 and HMC3 cells was applied to PC12 neuronal cells to evaluate cell viability and differentiation by immunofluorescence. In BV2 microglial cells, P. vulgatus LPS did not induce nitric oxide (NO) production or iNOS expression. In both BV2 and HMC3 cells, it failed to trigger pro-inflammatory cytokine release or TLR4 pathway activation. CM from E. coli-treated microglia disrupted MAP2 expression in PC12 neurons, whereas media from P. vulgatus-treated microglia did not. Overall, our data argue that “LPS-induced neuroinflammation” is not a universal phenomenon, but a chemistry-dependent outcome shaped by specific LPS structures. This study therefore highlights the need to consider LPS structural diversity in neuroinflammation models, particularly in the context of gut-brain communication.

Lipopolysaccharides (LPSs) from Gram-negative bacteria are traditionally viewed as potent "endotoxins" recognized by the immune system and capable of triggering robust inflammation. However, increasing evidence from gut commensals is dismantling this one-dimensional view. The gastrointestinal tract is indeed the major reservoir of LPSs, owing to the dense Gram-negative community inhabiting the small and large intestine, with total weight in healthy individuals estimated to exceed one gram. This necessarily means that the mere presence of LPSs cannot be directly linked to inflammation. Moreover, chronic exposure to low-potency or atypical LPSs can recalibrate innate immunity, fostering tolerance or, conversely, failing to provide adequate tonic stimulation and thereby predisposing the system to aberrant activation. Understanding this delicate balance and the structural and cellular mechanisms that sustain it, is essential to interpret the immunological impact of the gut LPSs in health and disease. In this Perspective, we highlight recent advances revealing the remarkable chemical diversity of commensal-derived LPSs and illustrate how subtle variations in LPS lipid A acylation and phosphorylation, core oligosaccharide architecture, O-antigen composition, and overall supramolecular organization profoundly rewire receptor usage and downstream immune outcomes. These insights underscore the enormous, still largely untapped potential of gut LPS chemistry to reveal unifying structural hallmarks that distinguish inflammatory, tolerogenic, and immunologically "tuned" features. Although fragments of this logic are beginning to emerge, a comprehensive framework remains urgently needed. Decoding the chemical language adopted by LPSs in the gut will be essential to reclassify LPSs not merely as dangerous molecules, but as a potential source of immunomodulators and as a blueprint for next-generation tools enabling precision control of host-microbe interactions.

Gram-negative bacteria feature an asymmetric outer membrane, composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) or lipooligosaccharides (LOS) in the outer leaflet, which underpins virulence and antibiotic resistance. Conventional LPS assays like the Limulus amebocyte lysate test face ethical and practical challenges, motivating the development of aptamer-based detection. Here, we probe the interaction of the LA27 DNA aptamer (Kd ≈ 46 nM) with biomimetic membranes incorporating LOS/LPS from Akkermansia muciniphila, Flavobacterium sp. Root935, and Paenalcaligenes hominis strains. Asymmetric supported lipid bilayers (SLBs) and symmetric large unilamellar vesicles (LUVs) were characterized by neutron reflectometry (NR), small-angle neutron scattering (SANS), and dynamic light scattering (DLS). NR confirmed preserved bilayer asymmetry and revealed that LA27 deeply penetrates Akkermansia LOS bilayers, evidenced by increased core hydration and structural disruption, whereas interactions with Flavobacterium LOS are moderate, and those with Paenalcaligenes LPS are confined to surface adhesion due to extensive LPS O-antigen chains. SANS and DLS of LUVs corroborated these modes: Akkermansia vesicles showed negligible changes in lamellar thickness and hydrodynamic radius, consistent with insertion, while those containing Flavobacterium LOS and Paenalcaligenes LPS exhibited significant increases in both size-related metrics, indicative of surface binding for those latter cases. These results elucidate how polysaccharide composition and supramolecular architecture of LOS/LPS govern aptamer affinity and specificity, providing a rigorous framework for nano-bio interface studies and laying the groundwork for selective, aptamer-based endotoxin sensing platforms.

Gut microbiota play a pivotal role in maintaining immune homeostasis, with Phocaeicola vulgatus emerging as a key commensal bacterium modulating host inflammatory responses. Central to this immunomodulatory effect is its lipopolysaccharide (LPS), whose core oligosaccharide region has been implicated in selective recognition by dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Here, we present the efficient synthesis of a diverse set of oligosaccharide fragments from the P. vulgatus LPS core, providing a molecular toolkit to investigate their recognition by DC-SIGN. This novel synthetic approach enabled the generation of structurally well-defined glycans, which were further characterized by using a multidisciplinary strategy combining NMR spectroscopy and computational studies. Ligand-based NMR and molecular simulations provided key insights into the structural features driving DC-SIGN binding, highlighting the role of specific glycan motifs. Given that these synthetic epitopes mimic bacterial glycan signatures, they offer a valuable platform for broader screening studies aimed at uncovering novel lectin-glycan interactions across different microbial species. This approach opens new avenues for exploring the intricate molecular basis of bacterial glycan recognition, expanding our understanding of host-microbe interactions, and driving the design of glycomimetic probes for immune modulation.

Siglec-7, an immune checkpoint receptor, has emerged as a promising target for cancer immunotherapy due to its involvement in the regulation of immune and inflammatory responses. However, while its participation in immunoediting and immune evasion is well established, understanding its biological context, relevant ligands, and associated signalling pathways remains limited. Understanding these aspects is crucial for the development of effective immunotherapies targeting Siglec-7. In this study, three expression constructs of Siglec-7 were designed, expressed, and characterised, including an analysis of the oligomeric state of its extracellular domain. The N-terminal V-set Ig carbohydrate recognition domain was also produced in an isotopically double-labelled (13C,15N) mammalian cell growth medium. Two stable constructs suitable for biophysical and structural studies were identified. These findings reveal the noncovalent dimerisation of Siglec-7, offering new insights into its possible ligand interactions, signal transduction mechanisms, or receptor/ligand clustering. The dimerisation of Siglec-7 may be essential to achieve multivalent, high-avidity interactions with glycoconjugates, which may result in enhanced or alternative signalling processes within the NK cell immune synapse. In addition, a detailed protocol for generating double-labelled Siglec-7 in HEK293 cells, which may apply to other proteins under similar conditions, was described. These findings contribute to a better understanding of the biophysical and structural properties of Siglec-7 and are key to the design of more precise and effective cancer immunotherapies targeting Siglec-7.

The immunological effects of lipopolysaccharides (LPSs) from gut microbiota remain poorly explored, overshadowed by the longstanding view of LPS as a prototypical pro-inflammatory molecule. Herein, we report the first comprehensive chemical and immunological characterization of LPS from Segatella copri DSM 18205, a prominent member of the human oral and intestinal microbiota. This LPS features a unique chemical architecture, including a mannose- and glucose-rich oligosaccharide (OS) and a highly heterogeneous, hypo-acylated lipid A domain, as elucidated by advanced mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Functionally, S. copri LPS displayed attenuated TLR4 activation and weak pro-inflammatory activity. Strikingly, high-dimensional cytometry by time-of-flight (CyTOF) revealed a selective preservation of CD14+CD16+ monocytes, immune subsets typically depleted by canonical enterobacterial LPSs. These findings identify S. copri LPS as a chemically and functionally distinct microbial signature, offering new insights into host-microbiota immune crosstalk and highlighting its potential for microbiome-informed immunomodulatory strategies.

Gram-negative bacterium Paenalcaligenes hominis, which is increasingly prevalent in elderly individuals, is associated with cognitive decline and gut-brain axis dysfunction. Here, we present a comprehensive structural characterization of P. hominis lipopolysaccharide (LPS), a key modulator of immune recognition and the main component of its outer membrane. Using a multidisciplinary approach combining chemical, spectroscopic, spectrometric, biophysical and computational methods, we unveil a unique O-antigen characterized by a trisaccharide repeating unit containing rhamnose and glucosamine, displaying nonstoichiometric O-acetylation and a terminal methylated rhamnose capping the saccharide chain. Furthermore, we disclose a short core oligosaccharide and a Lipid A composed of penta- to tetra-acylated species. Notably, this LPS exhibits reduced activation of Toll-Like Receptor-dependent signaling compared to the highly immunostimulatory Escherichia coli LPS and elicits a poor pro-inflammatory cytokine response. Moreover, P. hominis LPS exhibits selective binding to immune lectins such as Ficolin-3 and Galectin-4, as shown by the microarray assays. This raises the possibility that lectin-mediated recognition may represent an alternative route of immune engagement, which could help explain altered immune responses observed in elderly individuals. These findings provide a molecular basis for further exploring the role of P. hominis LPS in microbiota-induced immune modulation and its possible impact on age-related inflammatory and neurodegenerative conditions.

Polaribacter sp. SM1127, a cold-adapted marine Gram-negative bacterium isolated from Laminaria in Arctic waters, plays a crucial role in nutrient cycling and biopolymer degradation in cold environments. Additionally, its exopolysaccharide (EPS) exhibits promising biotechnological potential, including antioxidant and wound-healing properties. This study focuses on the isolation and characterization of lipid A, the glycolipid component of Polaribacter sp. SM1127 lipopolysaccharide (LPS), by bypassing full LPS extraction and working directly with the ethanol precipitation product containing both EPS and bacterial cells. Mass spectrometry analysis reveals significant structural heterogeneity in the lipid A, with variations in fatty acid chain length, branching, saturation, and hydroxylation. These features likely enable the bacterium to fine-tune its response to fluctuating temperatures or other cold-related environmental stresses, contributing to resilience in the Arctic Ocean ecosystem. Furthermore, immunological assays demonstrate that both LPS and EPS produced by Polaribacter sp. SM1127 induce weak Toll-like receptor 4 activation and, in general, poorly stimulate the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, compared to Escherichia coli LPS. These findings suggest their potential as immunomodulatory agents, like vaccine adjuvants.

Fusobacterium nucleatum (Fn) is a Gram-negative bacterium predominantly found in the human oral cavity, occasionally linked to systemic diseases, including colorectal cancer. Bacterial lipopolysaccharides (LPSs) represent one of the possible virulence factors contributing to and promoting disease progression. Fn LPS is recognized by Siglec-7, a sialic acid-binding inhibitory receptor expressed on immune cells and promising novel target for cancer immunotherapy. Through a combined approach of structural biology, biophysics, NMR, and computational methods, we explored the molecular basis of the interaction between Siglec-7 and the LPS fromF. nucleatum ssp polymorphum 10953, whose O-antigen contains peculiar sugars such as the neuraminic acid and the AAT (FucpNAc4N). We discovered a novel Siglec-7 binding epitope within the LPS O-antigen repeating unit, defined by its internal sialic acid and AAT residues. We propose a wing-like movement of the O-antigen, where Siglec-7 BC and CC' loops alternately engage the O-antigen edges within the binding site, with the BC loop forming more stable interactions. We uncover a novel Fn10953 immune evasion mechanism and highlight Siglec-7 and LPS as novel therapeutic targets for Fn-associated CRC, providing new avenues for intervention.

Lipopolysaccharides (LPSs) isolated from marine bacteria represent a valuable resource for biomedical innovation. Here, we report the first structural elucidation of the lipid A moiety and a preliminary immunological assessment of the full LPS from the marine Gram-negative Rheinheimera japonica KMM 9513T. Using MALDI-TOF mass spectrometry (MS) and tandem MS, we show that the lipid A from R. japonica KMM 9513T exhibits a heterogeneous architecture, composed of mono- and bis-phosphorylated tetra- and penta-acylated species with variations in the acyl chain length, saturation, branching, and positional isomerism. Functionally, the full LPS was found to be immunologically silent toward TLR4-mediated NF-κB activation in HEK-Blue™ hTLR4 cells and triggered only modest, dose-dependent responses in differentiated human THP-1 macrophages. Strikingly, the R. japonica LPS was able to antagonize E. coli LPS-induced TLR4 activation, even at low doses. Overall, this study uncovers a structurally and functionally atypical marine LPS with a dual profile, inactive towards TLR4 yet capable of modulating LPS-induced signaling. These findings offer a promising basis to consider R. japonica LPS as a source of structural inspiration for the design of synthetic derivatives with controlled immunological properties.

With an enormous potential in immunology and vaccinology, lipopolysaccharides (LPSs) are among the most extensively studied bacteria-derived molecules. LPS centered studies are countless, and their results reverberate in all areas of the life sciences, including chemistry, biology, genetics, biophysics, and medicine. Most of these research activities are focused on the LPS-induced immune response activation by means of Myeloid Differentiation protein-2/Toll Like Receptor 4 (MD-2/TLR4) complex, which currently is the most largely explored LPS sensing pathway. However, the enormous structural variability of LPS allows interactions with numerous other receptors involved in a wide range of equally important immunological scenarios. In this review, we explore these additional LPS recognition systems, which operate within interconnected signaling cascades, highlighting their role in maintaining physiological homeostasis and their involvement in the development of severe human diseases. Understanding these pathways, their interconnections, and the crosstalk between them and TLR4/MD-2 is essential for guiding the development of pharmacologically active molecules that could specifically modulate the inflammatory response, paving the way to new strategies for combating immune-mediated diseases and resistant infections.

Arabinogalactans (AG) from the Mycobacterium tuberculosis (Mtb) cell wall represent potential therapeutic agents against the notorious disease tuberculosis (TB). However, the synthetic access to these long, highly branched, and complex arabinogalactans remains a challenging task, hindering structure–activity relationship studies. Here, we report the chemical synthesis of arabinogalactan 92-mer 1 and shorter sequences 14-mer 2 , 30-mer 3 , and 50-mer 4 from M. tuberculosis cell envelope via an orthogonal one-pot glycosylation strategy based on glycosyl ortho -(1-phenylvinyl)benzoates, which avoids such issues as aglycone transfer inherent to one-pot assemblies based on thioglycosides. The synthetic route also features the following characteristics: 1) highly stereoselective construction of eight 1,2- cis -Ara f -(1→2) linkages via hydrogen-bond-mediated aglycone delivery strategy; 2) effective one-pot assembly of several linear and branched glycans by strategic utilizations of glycosyl N -phenyltrifluoroacetimidates, ortho -alkynylbenzoates, and ortho -(1-phenylvinyl)benzoates; 3) a one-pot and convergent [(7 × 2 + 7) × 2 + 50] assembly of arabinogalactan 92-mer with the simultaneous formations of six furanosidic bonds. Conformational analysis using molecular dynamics simulations and NMR spectroscopy, as well as immunological studies of synthetic arabinogalactans 1 – 4 in human cell models, revealed that the surface-exposed 30-mer 3 epitope induced only a modest NF-κB activation while preserving cell viability.

Gangliosides, sialylated glycosphingolipids abundant in the nervous system, play crucial roles in neurotransmission, interaction with regulatory proteins, cell-cell recognition, and signaling. Altered gangliosides expression has been correlated with pathological processes, including cancer, inflammatory disorders, and autoimmune diseases. Gangliosides are important endogenous ligands of Siglecs (Sialic acid-binding immunoglobulin-type lectins), I-type lectins mostly expressed by immune cells, that specifically recognize sialylated glycans. Siglec-7, an inhibitory immune receptor on human natural killer cells, represents a potential target for tumor immunotherapy. Notably, the expression of Siglec-7 ligands is high in various cancers, such as pancreatic cancer and melanoma and lead to tumor immune evasion. Siglec-7 binds the disialylated ganglioside GD3, a tumor-associated antigen overexpressed on cancer cells to suppress immune responses. Using a combination of structural biology techniques, including Nuclear Magnetic Resonance (NMR), biophysical, and computational methods, the binding of Siglec-7 to GD3 and Gb3 derivatives is investigated, revealing the importance of ligand conformation in modulating binding energetics and affinity. The greater flexibility of Gb3 derivatives appears to negatively impact binding entropy, leading to lower affinity compared to GD3. A thorough understanding of these interactions could contribute to elucidating molecular mechanisms of cancer immune evasion and facilitate the development of ganglioside-based diagnostic and therapeutic strategies for cancer.

Chimera-type galectin-3 (Gal-3) is a β-galactoside-binding protein containing a single conserved carbohydrate-recognition domain, crucial in fibrosis and carcinogenesis. Selenium-based Gal-3 inhibitors have emerged as promising therapeutic agents, particularly for treating neoplastic diseases. Among them, a seleno-digalactoside (SeDG) substituted with a benzyl group at position 3 of both saccharide residues (benzyl 3,3'-seleno-digalactoside, SeDG-Bn), attracted considerable attention for its selectivity and potent inhibitory efficacy against Gal-3. NMR spectroscopy and molecular dynamics simulations were combined to investigate the binding of SeDG-Bn to Gal-3 at the molecular level. This approach revealed the recognized epitope, the binding mode within Gal-3 binding pocket and enabled the generation of a 3D model of the complex. Our findings show that the presence of a single benzyl group establishes hydrophobic contacts with amino acids in Gal-3 β-sheets S2 and S3, crucially enhancing the binding affinity compared to unmodified SeDG. The digalactose backbone orientation in Gal-3 binding site is partially modified by the benzyl group with respect to complexes with lactosamine and SeDG. These results provide valuable insights into the design of more potent and selective inhibitors for Gal-3, potentially contributing to new therapeutic strategies for conditions such as cancer and fibrosis.

Siglecs, sialic-acid-binding immunoglobulin-like lectins, are key immune cell receptors that recognize sialic acid residues on cell surfaces. Pathogens and tumor cells exploit Siglecs to evade immune responses and modulate immunity, contributing significantly to infectious disease and cancer pathogenesis. Siglec-7, primarily expressed on natural killer (NK) cells, functions as an inhibitory receptor, tightly regulating the immune activity. This study investigates the interaction between Siglec-7 and the capsular polysaccharide (CPS) of Neisseria meningitidis serogroup Y (Men-Y), a bacterium whose sialylated CPS is critical for virulence. We demonstrate that Men-Y CPS binds to inhibitory Siglec-7, potentially dampening immune recognition. We employed a multifaceted approach, combining biochemical and biophysical techniques to dissect this interaction. Enzyme-linked immunosorbent assays (ELISAs) and fluorescence titrations quantified the binding specificity and affinity. Ligand- and protein-based nuclear magnetic resonance (NMR) spectroscopy, coupled with computational modeling, provides detailed molecular insights. We highlight the critical influence of the Men-Y CPS conformation and sialic acid presentation on Siglec-7 binding. The specific arrangement of α-2,6-linked sialic acids on the CPS is crucial for Siglec-7 binding, demonstrating the importance of the CPS 3D structure. Preliminary immunological assays using stimulated U937 cells (a promonocytic cell line) further support the immunomodulatory role of Siglec-7 mediated by Men-Y CPS. These results offer valuable insights into the development of targeted therapeutic strategies against bacterial infections.