X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

Atomic-Level Dissection of DC-SIGN Recognition of Bacteroides vulgatus LPS Epitopes.

The evaluation of Bacteroides vulgatus mpk (BVMPK) lipopolysaccharide (LPS) recognition by DC-SIGN, a key lectin in mediating immune homeostasis, has been here performed. A fine chemical dissection of BVMPK LPS components, attained by synthetic chemistry combined to spectroscopic, biophysical, and computational techniques, allowed to finely map the LPS epitopes recognized by DC-SIGN. Our findings reveal BVMPK's role in immune modulation via DC-SIGN, targeting both the LPS O-antigen and the core oligosaccharide. Furthermore, when framed within medical chemistry or drug design, our results could lead to the development of tailored molecules to benefit the hosts dealing with inflammatory diseases.

Molecular recognition of Escherichia coli R1-type core lipooligosaccharide by DC-SIGN.

Due to their ability to recognize carbohydrate structures, lectins emerged as potential receptors for bacterial lipopolysaccharides (LPS). Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. We contributed to fill this gap by unveiling the molecular basis of the interaction between the lipooligosaccharide of Escherichia coli and the dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN). Specifically, a combination of different techniques, including fluorescence microscopy, surface plasmon resonance, NMR spectroscopy, and computational studies, demonstrated that DC-SIGN binds to the purified deacylated R1 lipooligosaccharide mainly through the recognition of its outer core pentasaccharide, which acts as a crosslinker between two different tetrameric units of DC-SIGN. Our results contribute to a better understanding of DC-SIGN-LPS interaction and may support the development of pharmacological and immunostimulatory strategies for bacterial infections, prevention, and therapy.

Melanoma tumour-derived glycans hijack dendritic cell subsets through C-type lectin receptor binding.

Dendritic cell (DC) subsets play a crucial role in shaping anti-tumour immunity. Cancer escapes from the control immune system by hijacking DC functions. Yet, bases for such subversion are only partially understood. Tumour cells display aberrant glycan motifs on surface glycoproteins and glycolipids. Such carbohydrate patterns can be sensed by DCs through C-type lectin receptors (CLRs) that are critical to shape and orientate immune responses. We recently demonstrated that melanoma tumour cells harboured an aberrant 'glyco-code,' and that circulating and tumour-infiltrating DCs from melanoma patients displayed major perturbations in their CLR profiles. To decipher whether melanoma, through aberrant glycan patterns, may exploit CLR pathways to mislead DCs and evade immune control, we explored the impact of glycan motifs aberrantly found in melanoma (neoglycoproteins [NeoGP] functionalised with Gal, Man, GalNAc, s-Tn, fucose [Fuc] and GlcNAc residues) on features of human DC subsets (cDC2s, cDC1s and pDCs). We examined the ability of glycans to bind to purified DCs, and assessed their impact on DC basal properties and functional features using flow cytometry, confocal microscopy and multiplex secreted protein analysis. DC subsets differentially bound and internalised NeoGP depending on the nature of the glycan. Strikingly, Fuc directly remodelled the expression of activation markers and immune checkpoints, as well as the cytokine/chemokine secretion profile of DC subsets. NeoGP interfered with Toll like receptor (TLR)-signalling and pre-conditioned DCs to exhibit an altered response to subsequent TLR stimulation, dampening antitumor mediators while triggering pro-tumoral factors. We further demonstrated that DC subsets can bind NeoGP through CLRs, and identified GalNAc/MGL and s-Tn/ C-type lectin-like receptor 2 (CLEC2) as potential candidates. Moreover, DC dysfunction induced by tumour-associated carbohydrate molecules may be reversed by interfering with the glycan/CLR axis. These findings revealed the glycan/CLR axis as a promising checkpoint to exploit in order to reshape potent antitumor immunity while impeding immunosuppressive pathways triggered by aberrant tumour glycosylation patterns. This may rescue DCs from tumour hijacking and improve clinical success in cancer patients.

High-Mannose Oligosaccharide Hemimimetics that Recapitulate the Conformation and Binding Mode to Concanavalin A, DC-SIGN and Langerin.

The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.

Fluorinated Man9 as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR.

Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction. The introduction of tags into these complex oligosaccharides could overcome these problems and facilitate NMR studies. Here, we show the preparation of the Man9 of high mannose with some fluorine tags and the study of the interaction with its receptor, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This fluorinated ligand has allowed us to apply heteronuclear two-dimensional (2D) 1H,19F STD-TOCSYreF NMR experiments, using the initial slope approach, which has facilitated the analysis of the Man9/DC-SIGN interaction, unequivocally providing the binding epitope.

The unique 3D arrangement of macrophage galactose lectin enables Escherichia coli lipopolysaccharide recognition through two distinct interfaces.

Lipopolysaccharides are a hallmark of gram-negative bacteria, and their presence at the cell surface is key for bacterial integrity. As surface-exposed components, they are recognized by immunity C-type lectin receptors present on antigen-presenting cells. Human macrophage galactose lectin binds Escherichia coli surface that presents a specific glycan motif. Nevertheless, this high-affinity interaction occurs regardless of the integrity of its canonical calcium-dependent glycan-binding site. NMR of macrophage galactose-type lectin (MGL) carbohydrate recognition domain and complete extracellular domain revealed a glycan-binding site opposite to the canonical site. A model of trimeric macrophage galactose lectin was determined based on a combination of small-angle X-ray scattering and AlphaFold. A disulfide bond positions the carbohydrate recognition domain perpendicular to the coiled-coil domain. This unique configuration for a C-type lectin orients the six glycan sites of MGL in an ideal position to bind lipopolysaccharides at the bacterial surface with high avidity.

VEO-IBD NOX1 variant highlights a structural region essential for NOX/DUOX catalytic activity.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders that result from an inappropriate inflammatory response to the microbiota in genetically susceptible individuals, often triggered by environmental stressors. Part of this response is the persistent inflammation and tissue injury associated with deficiency or excess of reactive oxygen species (ROS). The NADPH oxidase NOX1 is highly expressed in the intestinal epithelium, and inactivating NOX1 missense mutations are considered a risk factor for developing very early onset IBD. Albeit NOX1 has been linked to wound healing and host defence, many questions remain about its role in intestinal homeostasis and acute inflammatory conditions. Here, we used in vivo imaging in combination with inhibitor studies and germ-free conditions to conclusively identify NOX1 as essential superoxide generator for microbiota-dependent peroxynitrite production in homeostasis and during early endotoxemia. NOX1 loss-of-function variants cannot support peroxynitrite production, suggesting that the gut barrier is persistently weakened in these patients. One of the loss-of-function NOX1 variants, NOX1 p. Asn122His, features replacement of an asparagine residue located in a highly conserved HxxxHxxN motif. Modelling the NOX1-p22phox complex revealed near the distal heme an internal pocket restricted by His119 and Asn122 that is part of the oxygen reduction site. Functional studies in several human NADPH oxidases show that substitution of asparagine with amino acids with larger side chains is not tolerated, while smaller side chains can support catalytic activity. Thus, we identified a previously unrecognized structural feature required for the electron transfer mechanism in human NADPH oxidases.

High-Density Lipoprotein function is modulated by the SARS-CoV-2 spike protein in a lipid-type dependent manner.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

Powerful Avidity with a Limited Valency for Virus-Attachment Blockers on DC-SIGN: Combining Chelation and Statistical Rebinding with Structural Plasticity of the Receptor.

The C-type lectin receptor DC-SIGN has been highlighted as the coreceptor for the spike protein of the SARS-CoV-2 virus. A multivalent glycomimetic ligand, Polyman26, has been found to inhibit DC-SIGN-dependent trans-infection of SARS-CoV-2. The molecular details underlying avidity generation in such systems remain poorly characterized. In an effort to dissect the contribution of the known multivalent effects - chelation, clustering, and statistical rebinding - we studied a series of dendrimer constructs related to Polyman26 with a rod core rationally designed to engage simultaneously two binding sites of the tetrameric DC-SIGN. Binding properties of these compounds have been studied with a range of biophysical techniques, including recently developed surface plasmon resonance oriented-surface methodology. Using molecular modeling we addressed, for the first time, the impact of the carbohydrate recognition domains' flexibility of the DC-SIGN tetramer on the compounds' avidity. We were able to gain deeper insight into the role of different binding modes, which in combination produce a construct with a nanomolar affinity despite a limited valency. This multifaceted experimental-theoretical approach provides detailed understanding of multivalent ligand/multimeric protein interactions which can lead to future predictions. This work opens the way to the development of new virus attachment blockers adapted to different C-type lectin receptors of viruses.

The melanoma tumor glyco-code impacts human dendritic cells' functionality and dictates clinical outcomes.

Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells triggering anti-tumor immune responses, but tumor cells exploit their versatility to subvert their functions. Tumor cells harbor unusual glycosylation patterns, which can be sensed through glycan-binding receptors (lectins) expressed by immune cells that are crucial for DCs to shape and orientate antitumor immunity. Yet, the global tumor glyco-code and its impact on immunity has not been explored in melanoma. To decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients' clinical outcome and DC subsets' functionality. Specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, tumor cells differentially impacting cytokine production by DCs harbored distinct glyco-profiles. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal displayed inhibitory impacts on cDC1s and pDCs. We further identified potential booster glycans for cDC1s and pDCs. Targeting specific glycans on melanoma tumor cells restored DCs' functionality. The tumor glyco-code was also linked to the nature of the immune infiltrate. This study unveils the impact of melanoma glycan patterns on immunity, and paves the way for innovative therapeutic options. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from tumor' hijacking to reshape antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation.

CRISPR-gene-engineered CYBB knock-out PLB-985 cells, a useful model to study functional impact of X-linked chronic granulomatous disease mutations: application to the G412E X91+-CGD mutation.

Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils.

Adenovirus-Inspired Virus-Like-Particles Displaying Melanoma Tumor Antigen Specifically Target Human DC Subsets and Trigger Antigen-Specific Immune Responses.

Virus-like particles constitute versatile vectors that can be used as vaccine platforms in many fields from infectiology and more recently to oncology. We previously designed non-infectious adenovirus-inspired 60-mer dodecahedric virus-like particles named ADDomers displaying on their surface either a short epitope or a large tumor/viral antigen. In this work, we explored for the first time the immunogenicity of ADDomers exhibiting melanoma-derived tumor antigen/epitope and their impact on the features of human dendritic cell (DC) subsets. We first demonstrated that ADDomers displaying tumor epitope/antigen elicit a strong immune-stimulating potential of human DC subsets (cDC2s, cDC1s, pDCs), which were able to internalize and cross-present tumor antigen, and subsequently cross-prime antigen-specific T-cell responses. To further limit off-target effects and enhance DC targeting, we engineered specific motifs to de-target epithelial cells and improve DCs' addressing. The improved engineered platform making it possible to display large antigen represents a tool to overcome the barrier of immune allele restriction, broadening the immune response, and paving the way to its potential utilization in humans as an off-the-shelf vaccine.

Mannobioside biomimetics that trigger DC-SIGN binding selectivity.

Selective DC-SIGN targeting vs. langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp2-iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.

Immobilization of Biantennary N-Glycans Leads to Branch Specific Epitope Recognition by LSECtin.

The molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcß1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Glycomimetic ligands block the interaction of SARS-CoV-2 spike protein with C-type lectin co-receptors.

The C-type lectin receptors DC-SIGN and L-SIGN bind to glycans on the SARS-CoV-2 spike glycoprotein and promote trans-infection of ACE2-expressing cells. We tested C2 triazole-modified mono- and pseudo-di-mannosides as inhibitors of DC/L-SIGN binding to a model mannosylated protein (Man-BSA) and to SARS-CoV2 spike, finding that they inhibit the interaction of both lectins with the spike glycoprotein in a Surface Plasmon Resonance (SPR) assay and are more potent than mannose by up to 36-fold (DC-SIGN) and 10-fold (L-SIGN). The molecules described here are the first known glycomimetic ligands of L-SIGN.

Identification of a two-component regulatory system involved in antimicrobial peptide resistance in Streptococcus pneumoniae.

Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae, a total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E. coli. After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.

Immunization with synthetic SARS-CoV-2 S glycoprotein virus-like particles protects macaques from infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused an ongoing global health crisis. Here, we present as a vaccine candidate synthetic SARS-CoV-2 spike (S) glycoprotein-coated lipid vesicles that resemble virus-like particles. Soluble S glycoprotein trimer stabilization by formaldehyde cross-linking introduces two major inter-protomer cross-links that keep all receptor-binding domains in the "down" conformation. Immunization of cynomolgus macaques with S coated onto lipid vesicles (S-LVs) induces high antibody titers with potent neutralizing activity against the vaccine strain, Alpha, Beta, and Gamma variants as well as T helper (Th)1 CD4+-biased T cell responses. Although anti-receptor-binding domain (RBD)-specific antibody responses are initially predominant, the third immunization boosts significant non-RBD antibody titers. Challenging vaccinated animals with SARS-CoV-2 shows a complete protection through sterilizing immunity, which correlates with the presence of nasopharyngeal anti-S immunoglobulin G (IgG) and IgA titers. Thus, the S-LV approach is an efficient and safe vaccine candidate based on a proven classical approach for further development and clinical testing.

Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin.

Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.