Role of sialic acid in brachyspira hyodysenteriae adhesion to pig colonic mucins

Infection with Brachyspira hyodysenteriae results in mucoid hemorrhagic diarrhea. This pathogen is associated with the colonic mucus layer, mainly composed of mucins. Infection regulates mucin O-glycosylation in the colon and increases mucin secretion as well as B. hyodysenteriae binding sites on mucins. Here, we analyzed potential mucin epitopes for B. hyodysenteriae adhesion in the colon, as well as the effect of colonic mucins on bacterial growth. Associations between B. hyodysenteriae binding to pig colonic mucins and mucin glycan data showed that B. hyodysenteriae binding was associated with the presence of N-glycolylneuraminic acid (NeuGc) on mucins. The role of sialic acid in B. hyodysenteriae adhesion was analyzed after the removal of sialic acid residues on the mucins by enzymatic treatment with sialidase A, which decreased bacterial binding to the mucins. The effect of pig colonic mucins on B. hyodysenteriae growth was determined in carbohydrate-free medium. B. hyodysenteriae growth increased in the presence of mucins from two out of five infected pigs, suggesting utilization of mucins as a carbon source for growth. Additionally, bacterial growth was enhanced by free sialic acid and N-acetylglucosamine. The results highlight a role of sialic acid as an adhesion epitope for B. hyodysenteriae interaction with colonic mucins. Furthermore, the mucin response and glycosylation changes exerted in the colon during B. hyodysenteriae infection result in a potentially favorable environment for pathogen growth in the intestinal mucus layer

Optimized differential TCDA (D-TCDA) with novel differential feed structure

Modern phased arrays require large instantaneous bandwidths, wide fields of view, and low profiles to conduct multiple functions. Many of these phased arrays rely on emerging high speed ADCs and advanced balanced transceivers. The benefits of balanced front-ends include improved linearity, dynamic range, isolation, and noise resilience. The application of a differential phased array in such a system removes extraneous losses caused by baluns, though the issue of feed-borne E-plane scan resonances must be considered. We address the E-plane scan resonance issue through an improved Balanced Wideband Impedance Transformer (BWIT) feed for the ultra-wideband (UWB) Tightly Coupled Dipole Array (TCDA). This BWIT feed has already demonstrated mitigated common-modes over a 3:1 bandwidth ratio while scanning to low angles. Here, our differential TCDA (D-TCDA) is developed for the L-C band (viz. 1.0 GHz to 6.1 GHz) with emphasis on resonance-free wide-angle scanning. Rigorous EM model and circuit analysis is included to verify the BWIT performance. Under a VSWR \u3c 3 definition, the improved array achieves a 6:1 impedance bandwidth ratio (BWR) with resonance-free scanning in all planes. An exception is the H-plane scanning at 60◦ where the VSWR \u3c 4. Array simulations are verified with measurements for an 8×8 single-polarized prototype

Low-Cost S-Band Reconfigurable Monopole/Patch Antenna for CubeSats

The development of reconfigurable antennas compatible with a CubeSat form factor can aid several space missions. Often, satellite missions require multiple wireless links with the same radio, but the design of such antennas is challenging due to the mechanical constraints and the limited power aboard a CubeSat. In this article, we present a unique reconfigurable antenna concept enabled by adhesive polyimide tapes. The presented antenna can switch from a conventional patch to a monopole-like antenna with minimal actuation complexity. This reconfiguration provides choices for polarization, pattern, and gain without use of active components for size, cost and power consumption reductions. The frequency of operation is S-band (2.4 GHz), and the antenna achieves S_{11} \u3c -10 dB for both reconfiguration states. Measurements compare well with simulations in both states

Porphyromonas gingivalis Produce Neutrophil Specific Chemoattractants Including Short Chain Fatty Acids

Neutrophil migration from blood to tissue-residing microbes is governed by a series of chemoattractant gradients of both endogenous and microbial origin. Periodontal disease is characterized by neutrophil accumulation in the gingival pocket, recruited by the subgingival biofilm consisting mainly of gram-negative, anaerobic and proteolytic species such as Porphyromonas gingivalis. The fact that neutrophils are the dominating cell type in the gingival pocket suggests that neutrophil-specific chemoattractants are released by subgingival bacteria, but characterization of chemoattractants released by subgingival biofilm species remains incomplete. In the present study we characterized small (< 3 kDa) soluble chemoattractants released by growing P. gingivalis, and show that these are selective for neutrophils. Most neutrophil chemoattractant receptors are expressed also by mononuclear phagocytes, the free fatty acid receptor 2 (FFAR2) being an exception. In agreement with the selective neutrophil recruitment, the chemotactic activity found in P. gingivalis supernatants was mediated in part by a mixture of short chain fatty acids (SCFAs) that are recognized by FFAR2, and other leukocytes (including monocytes) did not respond to SCFA stimulation. Although SCFAs, produced by bacterial fermentation of dietary fiber in the gut, has previously been shown to utilize FFAR2, our data demonstrate that the pronounced proteolytic metabolism employed by P. gingivalis (and likely also other subgingival biofilm bacteria associated with periodontal diseases) may result in the generation of SCFAs that attract neutrophils to the gingival pocket. This finding highlights the interaction between SCFAs and FFAR2 in the context of P. gingivalis colonization during periodontal disease, but may also have implications for other inflammatory pathologies involving proteolytic bacteria

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, andN- andO-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidicN-glycans and lack ofO-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complexN-andO-glycans with remarkably elongatedN-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function

Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14–30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography–mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators. View Full-TextpublishedVersio

Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow–derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors

Sputum protein N-glycosylation in cystic fibrosis: a sweet response to bacterial colonisation

Thesis by publication.Bibliography: leaves 253-268.Introduction -- Cystic fibrosis and bacterial colonisation define the sputum N-glycosylation phenotype -- Investigating the link between clinical CF-typed Pseudomonas aeruginosa isolates and PAO1 laboratory strain with pauci-mannosidic N-glycans -- Proteome and N-glycoproteome of Cystic Fibrosis sputum links the pauci-mannosidic N-glycans with abundant neutrophil proteins -- Conclusions and future directions.Cystic fibrosis (CF) is a prevalent autosomal recessive disease characterised by chronicinfection and inflammation in the lungs. The damaged lungs secrete excess mucus that isexpectorated as sputum. The lung mucus is the primary site of microbial infection, withPseudomonas aeruginosa as one of the major infecting pathogens in CF patients. Alteration inglycosylation of mucins (heavily O-glycosylated proteins) has been shown to play a role inbacterial binding to the mucus in the lungs of CF patients. However, there has been littlereported on the N-glycosylation of the protein complement of CF sputum.We have characterized the total secreted protein N-glycome of sputum derived from five CFpatients, two non-CF patients infected with pathogens (iNCF) and two pathogen free non-CF(NCF) patients. The glycosylation difference between CF and iNCF, compared to betweeniNCF and NCF, sputum separates the role played by the CF disease itself in regulating Nglycanexpression as distinct from the effects of bacterial colonisation. A significant increasein unusual and truncated pauci-mannosidic N-glycans and a decrease in complex and hybridN-glycans on the sputum proteins were observed in CF and iNCF sputum compared to NCFsputum, suggesting a common effect of infection.To address the link between the variation in CF sputum N-glycans and bacterial colonisation,four P. aeruginosa clinical strains (PASS1, PASS2, PASS3 and PASS4) were isolated fromsputum of CF patients. We showed that secreted bacterial exoglycosidases were not thecause of the trimming of the N-glycans to pauci-mannosidic structures and that the bacterialproteins do not carry pauci-mannose N-glycans. The presence of the pauci-mannose Nglycanson the sputum proteins were thus not directly of bacterial origin.Proteomic analysis revealed an up-regulation of proteins involved in the immune response(specifically neutrophil proteins) in bacterial colonised (CF and iNCF) compared with noninfected(NCF) sputum. State-of-art site-specific N-glycoproteomic analysis of CF, iNCF andNCF sputum proteins showed that the abundant proteins of neutrophil origin, such asmyeloperoxidase and azurocidin, carried these truncated pauci-mannosidic N-glycans.Whilst abundant knowledge regarding mucin O-glycosylation is available in the literature,this study provides new structural N-glycan and site-specific N-glycoproteome informationto aid in understanding the complex cellular and molecular environment of the CF affectedrespiratory tract.Mode of access: World wide web1 online resource (xi, 269 leaves diagrams, graphs, table

Host mucin glycosylation plays a role in bacterial adhesion in lungs of individuals with cystic fibrosis

Malfunction of the cell surface glycoprotein, cystic fibrosis transmembrane conductance regulator, is the molecular hallmark of cystic fibrosis (CF), causing salt imbalance across the lung epithelium and biochemical and biophysical alterations of the mucous secretion and airway surfaces. Abnormal glycosylation of both secreted and membrane-tethered airway mucins in CF hosts are reported by a substantial body of literature and correlates with bacterial infection and inflammation in CF airways, features that are linked to the CF pathology. It is established that Pseudomonas aeruginosa and other CF-typic bacteria use the altered host mucin glycosylation as receptors for adhesion by dedicated lectins and adhesins recognizing an array of the aberrantly expressed glycan determinants. This review aims to describe the aberrant mucin glycosylation phenotype observed in CF airways relative to the non-CF equivalent by summarizing the wealth of literature on this topic. The possible causes and effects of altered glycosylation in the respiratory system are discussed. Specific attention is given to the adhesion mechanisms of the opportunistic P. aeruginosa, which utilizes the molecular alterations of the lung to gain access to the normally sterile airways. Finally, the emerging glycosylation-based therapeutics that show promising potential for reducing bacterial infection in individuals with CF by molecular mimicry mechanisms are discussed.24 page(s