Search

Antibodies to the mycobacterial surface lipoglycan lipoarabinomannan (LAM) and its related capsular polysaccharide arabinomannan (AM) are increasingly important for investigations focused on both understanding mechanisms of protection against Mycobacterium tuberculosis (Mtb) and developing next-generation point-of-care tuberculosis (TB) diagnostics. We provide here an overview of the growing pipeline of monoclonal antibodies (mAbs) to LAM/AM. Old and new methodologies for their generation are reviewed and we outline and discuss their glycan epitope specificity and other features with implications for the TB field.

Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (Kd = 100–200 μM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2–dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2. Mass spectrometric profiling of a glycan library reveals that sialylated glycans, especially sialic acid-containing gangliosides, interact with the RBD of the SARS-CoV-2 spike protein and are involved in ACE2-dependent viral infection.

The surface polysacharide arabinomannan (AM) and related glycolipid lipoarabinomannan (LAM) play critical roles in tuberculosis pathogenesis. Human antibody responses to AM/LAM are heterogenous and knowledge of reactivity to specific glycan epitopes at the monoclonal level is limited, especially in individuals who can control M. tuberculosis infection. We generated human IgG mAbs to AM/LAM from B cells of two asymptomatic individuals exposed to or latently infected with M. tuberculosis. Here, we show that two of these mAbs have high affinity to AM/LAM, are non-competing, and recognize different glycan epitopes distinct from other anti-AM/LAM mAbs reported. Both mAbs recognize virulent M. tuberculosis and nontuberculous mycobacteria with marked differences, can be used for the detection of urinary LAM, and can detect M. tuberculosis and LAM in infected lungs. These mAbs enhance our understanding of the spectrum of antibodies to AM/LAM epitopes in humans and are valuable for tuberculosis diagnostic and research applications., Elise Ishida et al. generate human monoclonal antibodies that can selectively recognize specific oligosaccharide epitopes of the polysaccharides arabinomannan and lipoarabinomannan, which are critical for M. tuberculosis pathogenesis. The authors demonstrate the utility of these antibodies in both diagnostic and laboratory settings, making them important tools for M. tuberculosis research.

A route for preparing lipooligosaccharide (LOS) glycans from

We report the

International audience; Viruses are a heterogeneous ensemble of entities, all sharing the need for a suitable host to replicate. They are extremely diverse, varying in morphology, size, nature, and complexity of their genomic content. Typically, viruses use host-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins. Thus, the structure of the glycans on the viral proteins have, to date, typically been considered to mimick those of the host. However, the more recently discovered large and giant viruses differ from this paradigm. At least some of these viruses code for an (almost) autonomous glycosylation pathway. These viral genes include those that encode the production of activated sugars, glycosyltransferases, and other enzymes able to manipulate sugars at various levels. This review focuses on large and giant viruses that produce carbohydrate-processing enzymes. A brief description of those harboring these features at the genomic level will be discussed, followed by the achievements reached with regard to the elucidation of the glycan structures, the activity of the proteins able to manipulate sugars, and the organic synthesis of some of these virus-encoded glycans. During this progression, we will also comment on many of the challenging questions on this subject that remain to be addressed. CONTENTS AJ 4.2. Chlorovirus N-Glycans AJ 5. Conclusions and Future Perspectives AN Author Information

The central dogma of biology does not allow for the study of glycans using DNA sequencing. We report a liquid glycan array (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1,500 copies of glycans per phage. A LiGA is synthesized by acylation of the phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins, such as CD22, on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identify the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo.

The biosynthesis of lipopolysaccharide (LPS), a key immunomodulatory molecule produced by gram-negative bacteria, has been a topic of long-term interest. To date, the chemical probes used as tools to study LPS biosynthetic pathways have consisted primarily of small fragments of the larger structure (e.g., the O-chain repeating unit). While such compounds have helped to provide significant insight into many aspects of LPS assembly, understanding other aspects will require larger, more complex probes. For example, the molecular interactions between polymeric LPS biosynthetic intermediates and the proteins that transfer them across the inner and outer membrane remain largely unknown. We describe the synthesis of two lipid-linked polysaccharides, containing 11 and 27 monosaccharide residues, that are related to LPS O-chain biosynthesis in Escherichia coli O9a. This work has led not only to multi-milligram quantities of two biosynthetic probes, but also provided insights into challenges that must be overcome in the chemical synthesis of structurally-defined polysaccharides., The synthesis of lipid-linked polysaccharides containing 11 and 27 monosaccharides via a ‘frame-shift’ strategy is described. The work provides biosynthetic probes and highlights challenges in synthesizing structurally-defined polymeric glycans.

Avocado consumption is associated with numerous health benefits. Avocadyne is a terminally unsaturated, 17-carbon long acetogenin found almost exclusively within avocados with noted anti-leukemia and anti-viral properties. In this study, specific structural features such as the terminal triple bond, odd number of carbons, and stereochemistry are shown to be critical to its ability to suppress mitochondrial fatty acid oxidation and impart selective activity in vitro and in vivo. Together, this is the first study to conduct a structure-activity analysis on avocadyne and outline the chemical moieties critical to fatty acid oxidation suppression.

The arabinogalactan of Corynebacterianeae is a critical heteropolysaccharide that tethers outer membrane mycolic acids to peptidoglycan thus forming the characteristic cell wall core of these prokaryotes. An essential α-(1→5)-arabinosyltransferase, AftA, is responsible for the transfer of the first arabinofuranosyl (Araf) unit of the arabinan domain to the galactan backbone of arabinogalactan, but the number and precise position at which Araf residue(s) is/are added in mycobacteria remain ill-defined. Using membrane preparations from Mycobacterium smegmatis overexpressing aftA, farnesyl-phospho-arabinose as an Araf donor, and a series of synthetic galactan acceptors of various lengths, we here show that a single priming arabinosyl residue substitutes the C-5 position of a precisely positioned internal 6-linked galactofuranosyl residue of the galactan acceptors, irrespective of their length. This unexpected result suggests that, like the structurally related mycobacterial lipoarabinomannans, the arabinogalactan of mycobacteria may in fact harbor a single arabinan chain.

BACKGROUND. Inadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM). METHODS. In this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard. RESULTS. Three hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval [CI] 6.3%–18.0%), 53.2% (95% CI 43.9%–62.1%), and 66.7% (95% CI 57.5%–74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%–95.0%), 98.9% (95% CI 96.7%–99.6%), and 98.1% (95% CI 95.6%–99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively. CONCLUSION. Compared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM’s potential as a biomarker. Additional research is required to assess FujiLAM’s performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings. FUNDING. Global Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt für Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases.

Chloroviruses produce a capsid protein containing N-linked glycans differing in structure from those found in all other organisms. These species feature a core "hyper-branched" fucose residue in which every hydroxyl group is glycosylated. We describe the synthesis of a nonasaccharide from Paramecium bursaria chlorella virus 1, one of most complex chlorovirus N-glycans reported, using a "counterclockwise" strategy involving the sequential addition of trisaccharide, disaccharide, and monosaccharide motifs to a trisaccharide containing the core fucose residue.

We report a siloxane-protected donor (7) for the highly stereoselective formation of β-(2,3-cis)-xylulofuranosyl bonds. Glycosylation reactions with 7 gave >80% yields, and only β-xylulofuranosides were isolated in all cases. The utility of 7 for the synthesis of complex glycans was shown by its successful application to the preparation of the repeating unit from the lipopolysaccharide O-antigen of Yersinia enterocolitica serovars O:5/O:5,27. This structure is a pentasaccharide with two β-xylulofuranose residues; using 7, both were introduced simultaneously with excellent stereocontrol.

International audience; Despite the impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and /composition in response to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes in its structure, but no reports exist supportings this assumption. Herein, using [MS2] recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we identified found that the mycobacterial protein GlfH1 (Rv3096), which protein exhibits an exo-β-D-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents the a first step towards understanding the remodeling of mycobacterial AG.

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-β-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-A resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans. Structural characterization of WbbM, an enzyme involved in O2a-antigen biosynthesis in Klebsiella pneumoniae, reveals two unique active sites with galactopyranosyl- or galactofuranosyl-transferase activities for oligosaccharide polymerization.

A better understanding of all immune components involved in protecting against Mycobacterium tuberculosis infection is urgently needed to inform strategies for novel immunotherapy and tuberculosis (TB) vaccine development. Although cell-mediated immunity is critical, increasing evidence supports that antibodies also have a protective role against TB. Yet knowledge of protective antigens is limited. Analyzing sera from 97 US immigrants at various stages of M. tuberculosis infection, we showed protective in vitro and in vivo efficacy of polyclonal IgG against the M. tuberculosis capsular polysaccharide arabinomannan (AM). Using recently developed glycan arrays, we established that anti-AM IgG induced in natural infection is highly heterogeneous in its binding specificity and differs in both its reactivity to oligosaccharide motifs within AM and its functions in bacillus Calmette-Guérin vaccination and/or in controlled (latent) versus uncontrolled (TB) M. tuberculosis infection. We showed that anti-AM IgG from asymptomatic but not from diseased individuals was protective and provided data suggesting a potential role of IgG2 and specific AM oligosaccharides. Filling a gap in the current knowledge of protective antigens in humans, our data support the key role of the M. tuberculosis surface glycan AM and suggest the importance of targeting specific glycan epitopes within AM in antibody-mediated immunity against TB.

A one-pot strategy for functionalizing pyranoside 1,2

Bacterial surface polysaccharides are assembled by glycosyltransferase enzymes that typically use sugar nucleotide or polyprenyl-monophosphosugar activated donors. Characterized representatives exist for many monosaccharides but neither the donor nor the corresponding glycosyltransferases have been definitively identified for ribofuranose residues found in some polysaccharides. Klebsiella pneumoniae O-antigen polysaccharides provided prototypes to identify dual-domain ribofuranosyltransferase proteins catalyzing a two-step reaction sequence. Phosphoribosyl-5-phospho-D-ribosyl-α-1-diphosphate serves as the donor for a glycan acceptor-specific phosphoribosyl transferase (gPRT), and a more promiscuous phosphoribosyl-phosphatase (PRP) then removes the residual 5'-phosphate. The 2.5-Å resolution crystal structure of a dual-domain ribofuranosyltransferase ortholog from Thermobacillus composti revealed a PRP domain that conserves many features of the phosphatase members of the haloacid dehalogenase family, and a gPRT domain that diverges substantially from all previously characterized phosphoribosyl transferases. The gPRT represents a new glycosyltransferase fold conserved in the most abundant ribofuranosyltransferase family.

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity “ruler.” We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl–coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors., This work was supported by the MINECO/FEDER EU contracts BFU2016-77427-C2-2-R, BFU2017-92223-EXP, PID2019-105649RB-I00, Severo Ochoa Excellence Accreditation SEV-2016-0644; the Basque Government contract KK-2019/00076; NIH R01AI149297 (to M.E.G.); the MINECO/FEDER EU contract BFU-2015-68981-P and the Basque Government (IT1264-19) (to F.-X.C.); MINECO/FEDER EU contract RTI2018-099592-B-C21 (to F.C.); the Canadian Glycomics Network Grant SD-1 (to T.L.L.). European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 844905 (to B.T.); the Basque Government (to I.A.); CONACyT fellowship no. 291276 (to E.D.P.-P.).

The outer surfaces of mycobacteria, including the organism that causes tuberculosis, are decorated with an array of immunomodulatory glycans. Among these are lipooligosaccharides (LOSs), a class of molecules for which the function remains poorly understood. We describe the chemical synthesis of the glycan portion of a tridecasaccharide LOS from the opportunistic pathogen Mycobacterium kansasii. The target contains a number of unusual structural motifs that complicate its assembly and is the most complex mycobacterial LOS glycan to be synthesized to date when considering size and number of unique monosaccharides and glycosidic linkages. These studies not only provide a roadmap for the preparation of additional members of this family of glycans, but also provides a valuable probe for use in structure-activity relationship investigations.

Lipopolysaccharide is an important immunomodulatory and structural component found in the outer membrane of Gram-negative bacteria. The biosynthesis of this glycoconjugate proceeds by a highly conserved pathway and, as such, is an attractive target for antibiotic action. We highlight here recent work focused on understanding this pathway with an emphasis on new insights related to chain length control and transport across the inner and outer cell membranes.

The de novo asymmetric synthesis of all possible stereoisomers of two polyketide natural products, avocadyne, avocadene, and the saturated variant avocadane, is described. The stereodivergent synthesis of the 12 congeners is accomplished in 4-6 steps from an achiral acylpyruvate derivative, which, in turn, is prepared in five steps from commercially available materials. The approach uses, sequentially, a Noyori asymmetric reduction, a diastereoselective chelate- or directed reduction of a β-hydroxyketone, and an ester reduction to a primary alcohol.

Summary Background Most tuberculosis-related deaths in people with HIV could be prevented with earlier diagnosis and treatment. The only commercially available tuberculosis point-of-care test (Alere Determine TB LAM Ag [AlereLAM]) has suboptimal sensitivity, which restricts its use in clinical practice. The novel Fujifilm SILVAMP TB LAM (FujiLAM) assay has been developed to improve the sensitivity of AlereLAM. We assessed the diagnostic accuracy of the FujiLAM assay for the detection of tuberculosis in hospital inpatients with HIV compared with the AlereLAM assay. Methods For this diagnostic accuracy study, we assessed biobanked urine samples obtained from the FIND Specimen Bank and the University of Cape Town Biobank, which had been collected from hospital inpatients (aged ≥18 years) with HIV during three independent prospective cohort studies done at two South African hospitals. Urine samples were tested using FujiLAM and AlereLAM assays. The conduct and reporting of each test was done blind to other test results. The primary objective was to assess the diagnostic accuracy of FujiLAM compared with AlereLAM, against microbiological and composite reference standards (including clinical diagnoses). Findings Between April 18, 2018, and May 3, 2018, urine samples from 968 hospital inpatients with HIV were evaluated. The prevalence of microbiologically-confirmed tuberculosis was 62% and the median CD4 count was 86 cells per μL. Using the microbiological reference standard, the estimated sensitivity of FujiLAM was 70·4% (95% CI 53·0 to 83·1) compared with 42·3% (31·7 to 51·8) for AlereLAM (difference 28·1%) and the estimated specificity of FujiLAM was 90·8% (86·0 to 94·4) and 95·0% (87·7–98·8) for AlereLAM (difference −4·2%). Against the composite reference standard, the specificity of both assays was higher (95·7% [92·0 to 98·0] for FujiLAM vs 98·2% [95·7 to 99·6] for AlereLAM; difference −2·5%), but the sensitivity of both assays was lower (64·9% [50·1 to 76·7] for FujiLAM vs 38·2% [28·1 to 47·3] for AlereLAM; difference 26·7%). Interpretation In comparison to AlereLAM, FujiLAM offers superior diagnostic sensitivity, while maintaining specificity, and could transform rapid point-of-care tuberculosis diagnosis for hospital inpatients with HIV. The applicability of FujiLAM for settings of intended use requires prospective assessment. Funding Global Health Innovative Technology Fund, UK Department for International Development, Dutch Ministry of Foreign Affairs, Bill & Melinda Gates Foundation, German Federal Ministry of Education and Research, Australian Department of Foreign Affairs and Trade, Wellcome Trust, Department of Science and Technology and National Research Foundation of South Africa, and South African Medical Research Council.

Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-β-d- manno-oct-2-ulosonic acid (β-Kdo). The β-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for β-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.

Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral co-receptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.

Emerging evidence suggests that host glycans influence infection by SARS-CoV-2. Here, we reveal that the receptor-binding domain (RBD) of the spike (S)-protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (SA), with preference for the oligosaccharide of monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind the RBD. The monomeric affinities (Kd = 100-200 μM) of gangliosides for the RBD are similar to heparan sulfate, another negatively charged glycan ligand of the RBD proposed as a viral coreceptor. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to ACE2-expressing cells is decreased upon depleting cell surface SA level using three approaches: sialyltransferase inhibition, genetic knock-out of SA biosynthesis, or neuraminidase treatment. These effects on RBD binding and pseudotyped viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.