ABSTRACT
The bacterial genus Salmonella includes diverse isolates with multiple variations in the structure of the main polysaccharide component (O antigen) of membrane lipopolysaccharides. In addition, some isolates produce a transient (T) antigen, such as the T1 polysaccharide identified in the 1960s in an isolate of Salmonella enterica Paratyphi B. The structure and biosynthesis of the T1 antigen have remained enigmatic. Here, we use biophysical, biochemical and genetic methods to show that the T1 antigen is a complex linear glycan containing tandem homopolymeric domains of galactofuranose and ribofuranose, linked to lipid A-core, like a typical O antigen. T1 is a phase-variable antigen, regulated by recombinational inversion of the promoter upstream of the T1 genetic locus through a mechanism not observed for other bacterial O antigens. The T1 locus is conserved across many Salmonella isolates, but is mutated or absent in most typhoidal serovars and in serovar Enteritidis.
Subject(s)
O Antigens , O Antigens/genetics , O Antigens/metabolism , O Antigens/biosynthesis , Salmonella/genetics , Salmonella/metabolism , Gene Expression Regulation, Bacterial , Serogroup , Promoter Regions, Genetic , Polysaccharides, Bacterial/biosynthesis , Polysaccharides, Bacterial/metabolismABSTRACT
Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.
ABSTRACT
Klebsiella pneumoniae provides influential prototypes for lipopolysaccharide O antigen (OPS) biosynthesis in Gram-negative bacteria. Sequences of OPS-biosynthesis gene clusters in serotypes O4 and O7 suggest fundamental differences in the organization of required enzyme modules compared to other serotypes. Furthermore, some required activities were not assigned by homology shared with characterized enzymes. The goal of this study was therefore to resolve the serotype O4 and O7 pathways to expand our broader understanding of glycan polymerization and chain termination processes. The O4 and O7 antigens were produced from cloned genetic loci in recombinant Escherichia coli. Systematic in vivo and in vitro approaches were then applied to assign each enzyme in each of the pathways, defining the necessary components for polymerization and chain termination. OPS assembly is accomplished by multiprotein complexes formed by interactions between polymerase components variably distributed in single and multimodule proteins. In each complex, a terminator function is present in a protein containing a characteristic coiled-coil molecular ruler, which determines glycan chain length. In serotype O4, we discovered a CMP-α-3-deoxy-á´ -manno-octulosonic acid-dependent chain-terminating glycosyltransferase that is the founding member of a new glycosyltransferase family (GT137) and potentially identifies a new glycosyltransferase fold. The O7 OPS is terminated by a methylphosphate moiety, like the K. pneumoniae O3 antigen, but the methyltransferase-kinase enzyme pairs responsible for termination in these serotypes differ in sequence and predicted structures. Together, the characterization of O4 and O7 has established unique enzyme activities and provided new insight into glycan-assembly strategies that are widely distributed in bacteria.
Subject(s)
Bacterial Proteins , Klebsiella pneumoniae , O Antigens , Klebsiella pneumoniae/metabolism , Klebsiella pneumoniae/genetics , O Antigens/metabolism , O Antigens/genetics , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/genetics , Escherichia coli/metabolism , Escherichia coli/genetics , Multigene FamilyABSTRACT
Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.
Subject(s)
Glycosyltransferases , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Glycosylation , Citrobacter/metabolism , Citrobacter/genetics , O Antigens/metabolism , O Antigens/chemistry , Polysaccharides/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Polysaccharides, Bacterial/metabolismABSTRACT
Mycobacterium abscessus is increasingly recognized as the causative agent of chronic pulmonary infections in humans. One of the genes found to be under strong evolutionary pressure during adaptation of M. abscessus to the human lung is embC which encodes an arabinosyltransferase required for the biosynthesis of the cell envelope lipoglycan, lipoarabinomannan (LAM). To assess the impact of patient-derived embC mutations on the physiology and virulence of M. abscessus, mutations were introduced in the isogenic background of M. abscessus ATCC 19977 and the resulting strains probed for phenotypic changes in a variety of in vitro and host cell-based assays relevant to infection. We show that patient-derived mutational variations in EmbC result in an unexpectedly large number of changes in the physiology of M. abscessus, and its interactions with innate immune cells. Not only did the mutants produce previously unknown forms of LAM with a truncated arabinan domain and 3-linked oligomannoside chains, they also displayed significantly altered cording, sliding motility, and biofilm-forming capacities. The mutants further differed from wild-type M. abscessus in their ability to replicate and induce inflammatory responses in human monocyte-derived macrophages and epithelial cells. The fact that different embC mutations were associated with distinct physiologic and pathogenic outcomes indicates that structural alterations in LAM caused by nonsynonymous nucleotide polymorphisms in embC may be a rapid, one-step, way for M. abscessus to generate broad-spectrum diversity beneficial to survival within the heterogeneous and constantly evolving environment of the infected human airway.
Subject(s)
Mycobacterium abscessus , Humans , Bacterial Proteins/genetics , Lipopolysaccharides/chemistry , MutationABSTRACT
A deâ novo asymmetric strategy for the synthesis of d-bradyrhizose diastereomers from an achiral ketoenolester precursor is described. Key transformations used in the stereodivergent approach include two Noyori asymmetric reductions, an Achmatowicz rearrangement, diastereoselective alkene oxidations, and the first example of a palladium(0)-catalyzed glycosylation of a vinylogous pyranone. The isomeric composition of the bicyclic reducing sugars obtained was analyzed and their behaviour was compared to the natural product, revealing key stereocentres that impact the overall distribution.
ABSTRACT
Campylobacters are important causes of gastrointestinal illness and the capsular polysaccharides (CPS) they produce are key virulence factors and targets for vaccine development. We report here the synthesis of two fragments of the Campylobacter jejuni CG8486 strain CPS that contain a rare 6-deoxy-d-ido-heptopyranose residue and, in one target, two O-methyl phosphoramidate (MeOPN) motifs. The synthetic approach features the stereoselective construction of the ß-d-ido-heptopyranoside linkage via glycosylation with a ß-d-galacto-heptopyranoside donor followed by a one-pot sequential C-2 and C-3 inversion. During the syntheses, we uncovered a number of interesting conformational effects with regard to the 6-deoxy-ido-heptopyranose ring, the glycosidic linkage connecting the two monosaccharides, and the MeOPN groups.
Subject(s)
Campylobacter jejuni , Polysaccharides, Bacterial , Polysaccharides, Bacterial/chemistry , Campylobacter jejuni/chemistry , Campylobacter jejuni/metabolism , Monosaccharides , GlycosylationABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0274415.].
ABSTRACT
BACKGROUND: Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. METHODS: In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. FINDINGS: Macaques that developed LTBI (n = 36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n = 21; p < 0.01, q < 0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p < 0.05) and increasing airway IgG responses to some proteins over time. INTERPRETATION: Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. FUNDING: NIH/NIAID AI117927, AI146329, and AI127173 to JMA.
Subject(s)
Latent Tuberculosis , Mycobacterium tuberculosis , Tuberculosis , Animals , Antibody Formation , Antigens, Bacterial , Immunoglobulin G , Polysaccharides , Macaca , Primates , Immunoglobulin AABSTRACT
A better understanding of the epitopes most relevant for antibody-mediated protection against tuberculosis (TB) remains a major knowledge gap. We have shown that human polyclonal IgG against the Mycobacterium tuberculosis (M. tuberculosis) surface glycan arabinomannan (AM) and related lipoarabinomannan (LAM) is protective against TB. To investigate the impact of AM epitope recognition and Fcγ receptor (FcγR) binding on antibody functions against M. tuberculosis, we isolated a high-affinity human monoclonal antibody (mAb; P1AM25) against AM and showed its binding to oligosaccharide (OS) motifs we previously found to be associated with in vitro functions of human polyclonal anti-AM IgG. Human IgG1 P1AM25, but not 2 other high-affinity human IgG1 anti-AM mAbs reactive with different AM OS motifs, enhanced M. tuberculosis phagocytosis by macrophages and reduced intracellular growth in an FcγR-dependent manner. P1AM25 in murine IgG2a, but neither murine IgG1 nor a non-FcγR-binding IgG, given intraperitoneally prior to and after aerosolized M. tuberculosis infection, was protective in C57BL/6 mice. Moreover, we demonstrated the protective efficacy of human IgG1 P1AM25 in passive transfer with M. tuberculosis-infected FcγR-humanized mice. These data enhance our knowledge of the important interplay between both antibody epitope specificity and Fc effector functions in the defense against M. tuberculosis and could inform development of vaccines against TB.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Animals , Mice , Receptors, IgG/metabolism , Mice, Inbred C57BL , Tuberculosis/prevention & control , Antibodies, Monoclonal/pharmacology , Immunoglobulin G , EpitopesABSTRACT
Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis on phage to produce a genetically-encoded liquid glycan array (LiGA) of complex type N-glycans. Implementing the approach involved by ligating an azide-containing sialylglycosyl-asparagine to phage functionalized with 50-1000 copies of dibenzocyclooctyne. The resulting intermediate can be trimmed by glycosidases and extended by glycosyltransferases yielding a phage library with different N-glycans. Post-reaction analysis by MALDI-TOF MS allows rigorous characterization of N-glycan structure and mean density, which are both encoded in the phage DNA. Use of this LiGA with fifteen glycan-binding proteins, including CD22 or DC-SIGN on cells, reveals optimal structure/density combinations for recognition. Injection of the LiGA into mice identifies glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides a quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.
Subject(s)
Asparagine , Bacteriophages , Animals , Mice , Glycosylation , Azides , Gene LibraryABSTRACT
Carbapenemase and extended ß-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria.
Subject(s)
Klebsiella Infections , Klebsiella pneumoniae , Animals , Klebsiella pneumoniae/genetics , Lipopolysaccharides , O Antigens , Klebsiella , Blotting, Western , Klebsiella Infections/prevention & controlABSTRACT
Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.
Subject(s)
Extracellular Vesicles , Sialic Acid Binding Immunoglobulin-like Lectins , Female , Humans , Pregnancy , Extracellular Vesicles/metabolism , Glycolipids/chemistry , Glycolipids/metabolism , Liposomes , Mast Cells/metabolism , Memory B Cells/metabolism , Placenta/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolismABSTRACT
Enterocloster bolteae (formerly known as Clostridium bolteae) is a gastro-intestinal pathogenic bacterium often detected in the fecal microbiome of children in the autism spectrum. E. bolteae excretes metabolites that are thought to act as neurotoxins. This study is an update of our first E. bolteae investigation that discovered an immunogenic polysaccharide. Through a combination of chemical derivatizations/degradations, spectrometry and spectroscopy techniques, a polysaccharide composed of disaccharide repeating blocks comprised of 3-linked ß-d-ribofuranose and 4-linked α-l-rhamnopyranose, [â3)-ß-D-Ribf-(1 â 4)-α-L-Rhap-(1â]n, was identified. To confirm the structure, and to provide material for subsequent investigations, the chemical synthesis of a corresponding linker-equipped tetrasaccharide, ß-D-Ribf-(1 â 4)-α-L-Rhap-(1 â 3)-ß-D-Ribf-(1 â 4)-α-L-Rhap-(1âO(CH2)8N3, is also described. Research tools based on this immunogenic glycan structure can form the foundation for serotype classification, diagnostic/vaccine targets and clinical studies into the hypothesized role of E. bolteae in the onset/augmentation of autism related conditions in children.
Subject(s)
Autistic Disorder , Vaccines , Child , Humans , Autistic Disorder/microbiology , Clostridiales , OligosaccharidesABSTRACT
KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2â4) and ß-(2â7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2â4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.
Subject(s)
Bacterial Capsules , Glycosyltransferases , Bacterial Capsules/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Glycolipids/metabolism , Glycosyltransferases/genetics , Glycosyltransferases/chemistry , Lipopolysaccharides/metabolism , Sugar Acids/metabolism , Transferases/metabolism , Polysaccharides, Bacterial/metabolismABSTRACT
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.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Lipopolysaccharides , Antibodies, Monoclonal , Tuberculosis/microbiologyABSTRACT
WbbB, a lipopolysaccharide O-antigen synthesis enzyme from Raoultella terrigena, contains an N-terminal glycosyltransferase domain with a highly modified architecture that adds a terminal ß-Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) residue to the O-antigen saccharide, with retention of stereochemistry. We show, using mass spectrometry, that WbbB forms a covalent adduct between the catalytic nucleophile, Asp232, and Kdo. We also determine X-ray structures for the CMP-ß-Kdo donor complex, for Kdo-adducts with D232N and D232C WbbB variants, for a synthetic disaccharide acceptor complex, and for a ternary complex with both a Kdo-adduct and the acceptor. Together, these structures show that the enzyme-linked Asp232-Kdo adduct rotates to reposition the Kdo into a second sub-site, which then transfers Kdo to the acceptor. Retaining glycosyltransferases were thought to use only the front-side SNi substitution mechanism; here we show that retaining glycosyltransferases can also potentially use double-displacement mechanisms, but incorporating an additional catalytic subsite requires rearrangement of the protein's architecture.
Subject(s)
Glycosyltransferases , Lipopolysaccharides , Glycosyltransferases/genetics , Lipopolysaccharides/chemistry , O Antigens , Cytidine Monophosphate , DisaccharidesABSTRACT
A route for preparing lipooligosaccharide (LOS) glycans from Mycobacterium tuberculosis Canetti was developed and applied to the most complex of these structures, LOS II. The synthesis of the target nonasaccharide was achieved via a convergent [3+3+3] approach. Key features of the strategy include the stereoselective synthesis of an asymmetrically substituted trehalose moiety from two protected glucose residues and several chemoselective glycosylations involving thioglycoside donors.
Subject(s)
Mycobacterium tuberculosis , Lipopolysaccharides/chemistry , Mycobacterium , Mycobacterium tuberculosis/chemistry , Trehalose/chemistryABSTRACT
We report the de novo asymmetric synthesis of the 3,6-dideoxy sugars abequose, paratose, and tyvelose from 2-acetylfuran. Conversion of this readily available ketone to a pyranone derivative was followed by transformation to either an α- or ß-glycoside via diasteroselective acylation. Michael addition at C2 controlled primarily by the C1 configuration in the glycoside produced 3,6-dideoxy-4-keto sugars, which could be reduced and converted to either fully deprotected monosaccharides or to immediate precursors of glycosyl donors.