Extending the enzymatic toolbox for heparosan polymerization, depolymerization, and detection.

Heparosan is an acidic polysaccharide expressed as a capsule polymer by pathogenic and commensal bacteria, e.g. by E. coli K5. As a precursor in the biosynthesis of heparan sulfate and heparin, heparosan has a high biocompatibility and is thus of interest for pharmaceutical applications. However, due to its low immunogenicity, developing antibodies against heparosan and detecting the polymer in biological samples has been challenging. In this study, we exploited the enzyme repertoire of E. coli K5 and the E. coli K5-specific bacteriophage ΦK5B for the controlled synthesis and depolymerization of heparosan. A fluorescently labeled heparosan nonamer was used as a priming acceptor to study the elongation mechanism of the E. coli K5 heparosan polymerases KfiA and KfiC. We could demonstrate that the enzymes act in a distributive manner, producing labeled heparosan of low dispersity. The enzymatically synthesized heparosan was a useful tool to identify the tailspike protein KflB of ΦK5B as heparosan lyase and to characterize its endolytic depolymerization mechanism. Most importantly, using site-directed mutagenesis and rational construct design, we generated an inactive version of KflB for the detection of heparosan in ELISA-based assays, on blots, and on bacterial and mammalian cells.

A multi-enzyme machine polymerizes the Haemophilus influenzae type b capsule.

Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.

Rescue of synaptic and cognitive functions in polysialic acid-deficient mice and dementia models by short polysialic acid fragments.

Dysregulated cortical expression of the neural cell adhesion molecule (NCAM) and deficits of its associated polysialic acid (polySia) have been found in Alzheimer's disease and schizophrenia. However, the functional role of polySia in cortical synaptic plasticity remains poorly understood. Here, we show that acute enzymatic removal of polySia in medial prefrontal cortex (mPFC) slices leads to increased transmission mediated by the GluN1/GluN2B subtype of N-methyl-d-aspartate receptors (NMDARs), increased NMDAR-mediated extrasynaptic tonic currents, and impaired long-term potentiation (LTP). The latter could be fully rescued by pharmacological suppression of GluN1/GluN2B receptors, or by application of short soluble polySia fragments that inhibited opening of GluN1/GluN2B channels. These treatments and augmentation of synaptic NMDARs with the glycine transporter type 1 (GlyT1) inhibitor sarcosine also restored LTP in mice deficient in polysialyltransferase ST8SIA4. Furthermore, the impaired performance of polySia-deficient mice and two models of Alzheimer's disease in the mPFC-dependent cognitive tasks could be rescued by intranasal administration of polySia fragments. Our data demonstrate the essential role of polySia-NCAM in the balancing of signaling through synaptic/extrasynaptic NMDARs in mPFC and highlight the therapeutic potential of short polySia fragments to restrain GluN1/GluN2B-mediated signaling.

Sialic acids on B cells are crucial for their survival and provide protection against apoptosis.

Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas-deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

Mix-and-Match System for the Enzymatic Synthesis of Enantiopure Glycerol-3-Phosphate-Containing Capsule Polymer Backbones from Actinobacillus pleuropneumoniae, Neisseria meningitidis, and Bibersteinia trehalosi.

Capsule polymers are crucial virulence factors of pathogenic bacteria and are used as antigens in glycoconjugate vaccine formulations. Some Gram-negative pathogens express poly(glycosylglycerol phosphate) capsule polymers that resemble Gram-positive wall teichoic acids and are synthesized by TagF-like capsule polymerases. So far, the biotechnological use of these enzymes for vaccine developmental studies was restricted by the unavailability of enantiopure CDP-glycerol, one of the donor substrates required for polymer assembly. Here, we use CTP:glycerol-phosphate cytidylyltransferases (GCTs) and TagF-like polymerases to synthesize the poly(glycosylglycerol phosphate) capsule polymer backbones of the porcine pathogen Actinobacillus pleuropneumoniae, serotypes 3 and 7 (App3 and App7). GCT activity was confirmed by high-performance liquid chromatography, and polymers were analyzed using comprehensive nuclear magnetic resonance studies. Solid-phase synthesis protocols were established to allow potential scale-up of polymer production. In addition, one-pot reactions exploiting glycerol-kinase allowed us to start the reaction from inexpensive, widely available substrates. Finally, this study highlights that multidomain TagF-like polymerases can be transformed by mutagenesis of active site residues into single-action transferases, which in turn can act in trans to build-up structurally new polymers. Overall, our protocols provide enantiopure, nature-identical capsule polymer backbones from App2, App3, App7, App9, and App11, Neisseria meningitidis serogroup H, and Bibersteinia trehalosi serotypes T3 and T15. IMPORTANCE Economic synthesis platforms for the production of animal vaccines could help reduce the overuse and misuse of antibiotics in animal husbandry, which contributes greatly to the increase of antibiotic resistance. Here, we describe a highly versatile, easy-to-use mix-and-match toolbox for the generation of glycerol-phosphate-containing capsule polymers that can serve as antigens in glycoconjugate vaccines against Actinobacillus pleuropneumoniae and Bibersteinia trehalosi, two pathogens causing considerable economic loss in the swine, sheep, and cattle industries. We have established scalable protocols for the exploitation of a versatile enzymatic cascade with modular architecture, starting with the preparative-scale production of enantiopure CDP-glycerol, a precursor for a multitude of bacterial surface structures. Thereby, our approach not only allows the synthesis of capsule polymers but might also be exploitable for the (chemo)enzymatic synthesis of other glycerol-phosphate-containing structures such as Gram-positive wall teichoic acids or lipoteichoic acids.

Accelerated production of α2,8- and α2,9-linked polysialic acid in recombinant Escherichia coli using high cell density cultivation.

Polysialic acid (polySia) are α2,8- and/or α2,9-linked homopolymers with interesting properties for meningococcal vaccine development or the cure of human neurodegenerative disorders. With the goal to avoid large scale production of pathogenic bacteria, we compare in the current study the efficacy of conventional polySia production to recombinant approaches using the engineered laboratory safety strain E. coli BL21. High cell density cultivation (HCDC) experiments were performed in two different bioreactor systems. Increased cell densities of up to 11.3 (±0.4) g/L and polySia concentrations of up to 774 (±18) mg/L were reached in E. coli K1. However, cultivation of engineered E. coli BL21 strains delivered comparable cell densities but a maximum of only 133 mg/L polySia. Using established downstream procedures, host cell DNA and proteins were removed. All recombinant polySia products showed an identical degree of polymerization >90. Polymers with different glycosidic linkages could be successfully differentiated by nuclear magnetic resonance spectroscopy.

Differential expression patterns of glycosphingolipids and C-type lectin receptors on immune cells in absence of functional regulatory T cells.

BACKGROUND: Glycosylation is a common and complex type of protein posttranslational modification. Altered glycosylation of immunoglobulins in autoimmune diseases has led to the "altered glycan hypothesis" postulating existence of a unique glycan signature on immune cells and extracellular proteins characterized by site-specific relative abundances of individual glycan structures and glycosylation patterns. However, it is not clear how glycosylation on leukocyte subpopulations differ between states of health or inflammation. HYPOTHESIS: Glycosphingolipid patterns on immune cells of forkhead-box-P3-deficient scurfy mice differs from those on wild-type immune cells. METHODS: T cells and dendritic cells were isolated from spleens of either wild-type or age-matched scurfy mice. Glycosphingolipids of CD4+ T cells and splenic dendritic cells from wild-type and scurfy mice were then analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). In addition, flow cytometry and ChipCytometry were used to access expression patterns of various C-type lectin receptors on antigen-presenting cells from various organs of both wild-type and scurfy mice. RESULTS: We, hereby report differential expression of glycosphingolipids in health and under inflammatory conditions as reflected in wild-type and scurfy mice. Furthermore, we observed that the absence of functional regulatory T cells correlated with elevated expression of CLEC-7A and CD205 but a reduction in levels of CLEC12A and CD206 on antigen-presenting cells. CONCLUSION: We hereby show that the absence of functional regulatory T cells affects expression pattern and quantities of glycosphingolipids on immune cells. Thus, glycosphingolipids could serve as biomarkers for mapping genetical and homeostatic perturbances such as those resulting from a diseased condition.

An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-ß(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy.

Virus-neutralizing antibodies are a severe obstacle in oncolytic virotherapy. Here, we present a strategy to convert this unfavorable immune response into an anticancer immunotherapy via molecular retargeting. Application of a bifunctional adapter harboring a tumor-specific ligand and the adenovirus hexon domain DE1 for engaging antiadenoviral antibodies, attenuates tumor growth and prolongs survival in adenovirus-immunized mice. The therapeutic benefit achieved by tumor retargeting of antiviral antibodies is largely due to NK cell-mediated triggering of tumor-directed CD8 T-cells. We further demonstrate that antibody-retargeting (Ab-retargeting) is a feasible method to sensitize tumors to PD-1 immune checkpoint blockade. In therapeutic settings, Ab-retargeting greatly improves the outcome of intratumor application of an oncolytic adenovirus and facilitates long-term survival in treated animals when combined with PD-1 checkpoint inhibition. Tumor-directed retargeting of preexisting or virotherapy-induced antiviral antibodies therefore represents a promising strategy to fully exploit the immunotherapeutic potential of oncolytic virotherapy and checkpoint inhibition.

Approach for Profiling of Glycosphingolipid Glycosylation by Multiplexed Capillary Gel Electrophoresis Coupled to Laser-Induced Fluorescence Detection To Identify Cell-Surface Markers of Human Pluripotent Stem Cells and Derived Cardiomyocytes.

Application of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as tissue transplants in regenerative medicine depends on cell-surface marker-based characterization and/or purification. Glycosphingolipids (GSLs) are a family of highly diverse surface-exposed biomolecules that have been neglected as potential surface markers for hiPSC-CMs due to significant analytical challenges. Here, we describe the development of a novel and high-throughput-compatible workflow for the analysis of GSL-derived glycans based on ceramide glycanase digestion, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) labeling, and multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF). GSL glycans were detected with highly reproducible migration times after repeated analysis by xCGE-LIF. We built up a migration time database comprising 38 different glycan species, and we showed exemplarily that as few as 10 pg of fucosyl lactotetra was detectable. GSL glycan profiling could be performed with 105 human induced pluripotent stem cells, and we quantitatively dissected global alterations of GSL glycosylation of human induced pluripotent stem cells (hiPSCs) and hiPSC-CMs by employing xCGE-LIF. In our study, we observed a general switch from complex GSLs with lacto- and globo-series core structures comprising the well-known human pluripotent stem cell marker stage-specific embryonic antigen 3 (SSEA3) and SSEA4 in hiPSCs toward the simple gangliosides GM3 and GD3 in hiPSC-CMs. This is the first description of GM3 and GD3 being highly abundant GSLs on the cell surface of stem cell-derived cardiomyocytes.

Sialic acid is a critical fetal defense against maternal complement attack.

The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP-sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas-/- implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert's membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas-/- mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.

A patient-specific induced pluripotent stem cell model for West syndrome caused by ST3GAL3 deficiency.

ST3GAL3 encodes the Golgi enzyme beta-galactoside-alpha-2,3-sialyltransferase-III that in humans forms, among others, the sialyl Lewis a (sLea) epitope on proteins. Functionally deleterious variants in this gene were previously identified in patients with either non-syndromic or syndromic intellectual disability such as West syndrome, an age-dependent epileptic encephalopathic syndrome associated with developmental arrest or regression. The aim of this study was to further elucidate the molecular and cellular mechanisms causing West syndrome by lack of ST3GAL3 function. For this purpose we generated induced pluripotent stem cell (iPSC) lines from fibroblasts obtained from a patient with West syndrome, carrying a variant in exon 12 (c.958G>C, p.(Ala320Pro)) of ST3GAL3, and a healthy sibling, using lentiviral reprogramming. iPSCs and cortical neurons derived thereof were analysed by lectin blots, mRNA sequencing, adherence assays, and FACS. While no significant difference was observed at stem cell or fibroblast level between patient and control cells, patient-derived cortical neurons displayed an altered lectin blot staining pattern, enhanced adherence to a poly-L-ornithine/laminin-coated surface and decreased levels of neurons expressing T-box transcription factor brain 1. Our results suggest that changes in the sialylation pattern on the surface of specific neuronal cell types affect adhesive interactions during development, which in turn may cause subtle changes in tissue composition that could result in the occurrence of epilepsy and might impair neural development to an extent that is detrimental to the development and maintenance of normal cognitive functions.

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens.

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Bibersteinia trehalosi, and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis.IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy. The new capsule polymerase family described here vastly increases the toolbox of enzymes available for biotechnology purposes. Representatives are abundantly found in human pathogens but also in animal pathogens, paving the way for the exploitation of polymerases for the development of a new generation of vaccines for animal husbandry.

A single-step transconjugation system for gene deletion in Aggregatibacter actinomycetemcomitans.

Aggregatibacter (A.) actinomycetemcomitans is a periodontopathogenic bacterium causing aggressive periodontitis. Here we describe a single-step transconjugation system as novel and easily applicable protocol for site-specific genetic manipulation of A. actinomycetemcomitans. Deletion of PgaC, which is involved in the synthesis of biofilm matrix, led to a reduced biofilm formation.

Combined Chemical Synthesis and Tailored Enzymatic Elongation Provide Fully Synthetic and Conjugation-Ready Neisseria meningitidis Serogroup X Vaccine Antigens.

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.

Targeting polysialic acid-abundant cancers using oncolytic adenoviruses with fibers fused to active bacteriophage borne endosialidase.

Genetic replacement of adenoviral fiber knobs by ligands that enable tumor specific targeting of oncolytic adenoviruses is challenging because the fiber knob contributes to virus assembly. Here, we present a novel concept by describing stable recombinant adenoviruses with tumor specific infection mode. The fiber knob was replaced by endosialidaseNF (endoNF), the tailspike protein of bacteriophage K1F. EndoNF recognizes polysialic acid, an oncofetal antigen characteristic for high malignant tumors of neuroendocrine origin. An intramolecular chaperone contained in endoNF warrants folding and compensates for the knob function in virus assembly. Obtained recombinant viruses demonstrated polysialic acid dependent infection modes, strong oncolytic capacity with polysialic acid positive cells in culture and a high potential to inhibit tumor growth in a therapeutic mouse model of subcutaneous neuroblastoma. With a single genetic manipulation we achieved ablation of the fiber knob, introduction of a tumor specific ligand, and folding control over the chimeric fiber construct.

Efficient solid-phase synthesis of meningococcal capsular oligosaccharides enables simple and fast chemoenzymatic vaccine production.

Neisseria meningitidis serogroups A and X are among the leading causes of bacterial meningitis in the African meningitis belt. Glycoconjugate vaccines, consisting of an antigenic carrier protein coupled to the capsular polysaccharide of the bacterial pathogen, are the most effective strategy for prevention of meningococcal disease. However, the distribution of effective glycoconjugate vaccines in this region is limited by the high cost of cultivating pathogens and purification of their capsular polysaccharides. Moreover, chemical approaches to synthesize oligosaccharide antigens have proven challenging. In the current study, we present a chemoenzymatic approach for generating tailored oligosaccharide fractions ready for activation and coupling to the carrier protein. In a first step, the elongation modes of recombinant capsular polymerases from Neisseria meningitidis serogroups A (CsaB) and X (CsxA) were characterized. We observed that CsaB is a distributive enzyme, and CsxA is a processive enzyme. Sequence comparison of these two stealth family proteins revealed a C-terminal extension in CsxA, which conferred processivity because of the existence of a second product-binding site. Deletion of the C-terminal domain converted CsxA into a distributive enzyme, allowing facile control of product length by adjusting the ratio of donor to acceptor sugars. Solid-phase fixation of the engineered capsular polymerases enabled rapid production of capsular polysaccharides with high yield and purity. In summary, the tools developed here provide critical steps toward reducing the cost of conjugate vaccine production, which will increase access in regions with the greatest need. Our work also facilitates efforts to study the relationship between oligosaccharide size and antigenicity.

A sweet development in Notch regulation.

The transmembrane signaling protein Notch, which is crucial for embryonic cell fate decisions, has 36 extracellular EGF domains that are glycosylated in variable and complex ways. A new study shows that O-fucose and O-glucose stabilize the repeats but that extension of glucose by xylose weakens stability, explained by the binding of the glycan to a protein groove. This work shows how different types of glycosylation can distinctly influence protein stability and structure.