Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Immunoglobulin G N-glycan markers of accelerated biological aging during chronic HIV infection.

People living with HIV (PLWH) experience increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors associated with this vulnerability remain uncertain. In the general population, alterations in the N-glycans on IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgG N-glycans in cross-sectional and longitudinal samples from 1214 women and men, living with and without HIV. PLWH exhibit an accelerated accumulation of pro-aging-associated glycan alterations and heightened expression of senescence-associated glycan-degrading enzymes compared to controls. These alterations correlate with elevated markers of inflammation and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit a reduced ability to elicit anti-HIV Fc-mediated immune activities. These findings hold potential for the development of biomarkers and tools to identify and prevent premature aging and comorbidities in PLWH.

N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Galß-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

A weaponized phage suppresses competitors in historical and modern metapopulations of pathogenic bacteria.

Bacteriophages, the viruses of bacteria, are proposed to drive bacterial population dynamics, yet direct evidence of their impact on natural populations is limited. Here we identified viral sequences in a metapopulation of wild plant-associated Pseudomonas spp. genomes. We discovered that the most abundant viral cluster does not encode an intact phage but instead encodes a tailocin - a phage-derived element that bacteria use to kill competitors for interbacterial warfare. Each pathogenic Pseudomonas sp. strain carries one of a few distinct tailocin variants, which target variable polysaccharides in the outer membrane of co-occurring pathogenic strains. Analysis of historic herbarium samples from the last 170 years revealed that the same tailocin and receptor variants have persisted in the Pseudomonas populations for at least two centuries, suggesting the continued use of a defined set of tailocin haplotypes and receptors. These results indicate that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control. One-Sentence Summary: Bacterial pathogens in a host-associated metapopulation use a repurposed prophage to kill their competitors.

Pectic-AGP is a major form of Arabidopsis AGPs.

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca2+ signaling pathway as a Ca2+-capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. This result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

Low-level resource partitioning supports coexistence among functionally redundant bacteria during successional dynamics.

Members of microbial communities can substantially overlap in substrate use. However, what enables functionally redundant microorganisms to coassemble or even stably coexist remains poorly understood. Here, we show that during unstable successional dynamics on complex, natural organic matter, functionally redundant bacteria can coexist by partitioning low-concentration substrates even though they compete for one simple, dominant substrate. We allowed ocean microbial communities to self-assemble on leachates of the brown seaweed Fucus vesiculosus and then analyzed the competition among 10 taxonomically diverse isolates representing two distinct stages of the succession. All, but two isolates, exhibited an average of 90% ± 6% pairwise overlap in resource use, and functional redundancy of isolates from the same assembly stage was higher than that from between assembly stages, leading us to construct a simpler four-isolate community with two isolates from each of the early and late stages. We found that, although the short-term dynamics of the four-isolate communities in F. vesiculosus leachate was dependent on initial isolate ratios, in the long term, the four isolates stably coexist in F. vesiculosus leachate, albeit with some strains at low abundance. We therefore explored the potential for nonredundant substrate use by genomic content analysis and RNA expression patterns. This analysis revealed that the four isolates mainly differed in peripheral metabolic pathways, such as the ability to degrade pyrimidine, leucine, and tyrosine, as well as aromatic substrates. These results highlight the importance of fine-scale differences in metabolic strategies for supporting the frequently observed coexistence of large numbers of rare organisms in natural microbiomes.

Molecular-level architecture of Chlamydomonas reinhardtii's glycoprotein-rich cell wall.

Microalgae are a renewable and promising biomass for large-scale biofuel, food and nutrient production. However, their efficient exploitation depends on our knowledge of the cell wall composition and organization as it can limit access to high-value molecules. Here we provide an atomic-level model of the non-crystalline and water-insoluble glycoprotein-rich cell wall of Chlamydomonas reinhardtii. Using in situ solid-state and sensitivity-enhanced nuclear magnetic resonance, we reveal unprecedented details on the protein and carbohydrate composition and their nanoscale heterogeneity, as well as the presence of spatially segregated protein- and glycan-rich regions with different dynamics and hydration levels. We show that mannose-rich lower-molecular-weight proteins likely contribute to the cell wall cohesion by binding to high-molecular weight protein components, and that water provides plasticity to the cell-wall architecture. The structural insight exemplifies strategies used by nature to form cell walls devoid of cellulose or other glycan polymers.

Association of four differently processed diets with plasma and urine advanced glycation end products and serum soluble receptor for advanced glycation end products concentration in healthy dogs.

Advanced glycation end products (AGEs), formed via the Maillard reaction (MR) during processing of foods, have been implicated in inflammatory and degenerative diseases in human beings. Cellular damage is primarily caused by AGE binding with the receptor for AGEs (RAGE) on cell membranes. An isoform of RAGE, soluble RAGE (sRAGE), acts as a decoy receptor binding circulating AGEs preventing cellular activation. Pet food manufacturing involves processing methods similar to human food processing that may increase dietary AGEs (dAGEs). We hypothesized that diet, plasma and urine AGEs, and serum sRAGE concentrations would differ between thermally processed diets. This study examined the association of four differently processed diets: ultra-processed canned wet food (WF); ultra-processed dry food (DF); moderately processed air-dried food (ADF) and minimally processed mildly cooked food (MF) on total plasma levels of the AGEs, carboxymethyllysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1, glyoxal hydroimidazolone-1, argpyrimidine, urine CML, CEL and lysinoalanine, and serum sRAGE concentration. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used to measure AGEs. sRAGE concentration was measured using a commercial canine-specific enzyme-linked immunosorbent assay kit. Total dAGEs (mg/100 kcal as fed) were higher in WF than in other diets. Plasma total AGEs (nM/50 µL) were significantly higher with WF, with no difference found between DF, ADF, and MF; however, ADF was significantly higher than MF. Urine CML (nmol AGEs/mmol creatinine) was significantly higher with DF than with WF and MF. There were no significant differences in total urine AGEs or serum sRAGE concentration between diets. In conclusion, different methods of processing pet foods are associated with varied quantities of AGEs influencing total plasma AGE concentration in healthy dogs. Serum sRAGE concentration did not vary across diets but differences in total AGE/sRAGE ratio were observed between MF and WF and, ADF and DF.

Post-translational modification by the Pgf glycosylation machinery modulates Streptococcus mutans OMZ175 physiology and virulence.

Streptococcus mutans is commonly associated with dental caries and the ability to form biofilms is essential for its pathogenicity. We recently identified the Pgf glycosylation machinery of S. mutans, responsible for the post-translational modification of the surface-associated adhesins Cnm and WapA. Since the four-gene pgf operon (pgfS-pgfM1-pgfE-pgfM2) is part of the S. mutans core genome, we hypothesized that the scope of the Pgf system goes beyond Cnm and WapA glycosylation. In silico analyses and tunicamycin sensitivity assays suggested a functional overlap between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Phenotypic characterization of pgf mutants (ΔpgfS, ΔpgfE, ΔpgfM1, ΔpgfM2, and Δpgf) revealed that the Pgf system is important for biofilm formation, surface charge, membrane stability, and survival in human saliva. Moreover, deletion of the entire pgf operon (Δpgf strain) resulted in significantly impaired colonization in a rat oral colonization model. Using Cnm as a model, we showed that Cnm is heavily modified with N-acetyl hexosamines but it becomes heavily phosphorylated with the inactivation of the PgfS glycosyltransferase, suggesting a crosstalk between these two post-translational modification mechanisms. Our results revealed that the Pgf machinery contributes to multiple aspects of S. mutans pathobiology that may go beyond Cnm and WapA glycosylation.

A novel exopolysaccharide pathway from a freshwater Sphingomonas isolate.

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. To better understand the breadth of polysaccharide production in nature we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming that LM7 assembles a novel wzy/wzx-dependent polysaccharide. We extracted EPS from LM7 cultures and showed that it contains a linear chain of 3- and 4- linked glucose, galactose, and glucuronic acid residues. Finally, we found that the EPS pathway we identified diverges from those of adhesive polysaccharides such as the holdfast that are conserved in higher Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering of polysaccharides with valuable properties.

Reduction in N-Acetylglucosaminyltransferase-I Activity Decreases Survivability and Delays Development of Zebrafish.

A lack of complex and hybrid types of N-glycans in mice is embryonically lethal due to neural tube maldevelopment. N-acetylglucosaminyltransferase-I (GnT-I; Mgat1) catalyzes a required step for converting oligomannose N-glycans into hybrid and complex N-glycans. Unlike mice, zebrafish have two Mgat1a/b genes. Herein, CRISPR/Cas9 technology was used to knockdown GnT-Ib activity in zebrafish, referred to as Mgat1b-/-, to examine the impact of a decrease in complex types of N-glycans on survival and development, and sensory and motor functions. Genotyping verified the occurrence of edited Mgat1b, and LC-ESI-MS and lectin blotting identified higher levels of oligomannose and lower levels of complex N-glycans in Mgat1b-/- relative to Wt AB. The microscopic visualization of developmental stages and locomotor studies using an automated tracking unit and manual touch assays revealed reduced survivability, and delayed motor and sensory functions in Mgat1b-/-. Moreover, embryonic staging linked reduced survivability of Mgat1b-/- to disruption in brain anlagen formation. Birefringence measurements supported delayed skeletal muscle development, which corresponded with motor and sensory function impediments in Mgat1b-/-. Furthermore, GnT-Ib knockdown hindered cardiac activity onset. Collectively, Mgat1b-/- displayed incomplete penetrance and variable expressivity, such that some died in early embryonic development, while others survived to adulthood, albeit, with developmental delays. Thus, the results reveal that reducing the amount of complex-type N-glycans is unfavorable for zebrafish survival and development. Moreover, our results support a better understanding of human congenital disorders of glycosylation.

Campylobacter jejuni uses energy taxis and a dehydrogenase enzyme for l-fucose chemotaxis.

IMPORTANCE: In this study, we identify a separate role for the Campylobacter jejuni l-fucose dehydrogenase in l-fucose chemotaxis and demonstrate that this mechanism is not only limited to C. jejuni but is also present in Burkholderia multivorans. We now hypothesize that l-fucose energy taxis may contribute to the reduction of l-fucose-metabolizing strains of C. jejuni from the gastrointestinal tract of breastfed infants, selecting for isolates with increased colonization potential.

Knockout of the intellectual disability-linked gene Hs6st2 in mice decreases heparan sulfate 6-O-sulfation, impairs dendritic spines of hippocampal neurons, and affects memory.

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders.

Permethylation as a strategy for high-molecular-weight polysaccharide structure analysis by nuclear magnetic resonance-Case study of Xylella fastidiosa extracellular polysaccharide.

Current practices for structural analysis of extremely large-molecular-weight polysaccharides via solution-state nuclear magnetic resonance (NMR) spectroscopy incorporate partial depolymerization protocols that enable polysaccharide solubilization in suitable solvents. Non-specific depolymerization techniques utilized for glycosidic bond cleavage, such as chemical degradation or ultrasonication, potentially generate structural fragments that can complicate complete and accurate characterization of polysaccharide structures. Utilization of appropriate enzymes for polysaccharide degradation, on the other hand, requires prior structural knowledge and optimal enzyme activity conditions that are not available to an analyst working with novel or unknown compounds. Herein, we describe an application of a permethylation strategy that allows the complete dissolution of intact polysaccharides for NMR structural characterization. This approach is utilized for NMR analysis of Xylella fastidiosa extracellular polysaccharide (EPS), which is essential for the virulence of the plant pathogen that affects multiple commercial crops and is responsible for multibillion dollar losses each year.

Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion.

IMPORTANCE: The Gram-negative coccobacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by M. haemolytica are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation of neuA results in increased phagocytosis and complement-mediated killing of M. haemolytica, thus demonstrating that sialylation contributes to the virulence of M. haemolytica.

Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

IMPORTANCE: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Xylella fastidiosa modulates exopolysaccharide polymer length and the dynamics of biofilm development with a ß-1,4-endoglucanase.

IMPORTANCE: It is well established that exopolysaccharide (EPS) is an integral structural component of bacterial biofilms necessary for assembly and maintenance of the three-dimensional architecture of the biofilm. However, the process and role of EPS turnover within a developing biofilm is not fully understood. Here, we demonstrated that Xylella fastidiosa uses a self-produced endoglucanase to enzymatically process its own EPS to modulate EPS polymer length. This enzymatic processing of EPS dictates the early stages of X. fastidiosa's biofilm development, which, in turn, affects its behavior in planta. A deletion mutant that cannot produce the endoglucanase was hypervirulent, thereby linking enzymatic processing of EPS to attenuation of virulence in symptomatic hosts, which may be a vestige of X. fastidiosa's commensal behavior in many of its other non-symptomatic hosts.

Comparative mucomic analysis of three functionally distinct Cornu aspersum Secretions.

Every animal secretes mucus, placing them among the most diverse biological materials. Mucus hydrogels are complex mixtures of water, ions, carbohydrates, and proteins. Uncertainty surrounding their composition and how interactions between components contribute to mucus function complicates efforts to exploit their properties. There is substantial interest in commercializing mucus from the garden snail, Cornu aspersum, for skincare, drug delivery, tissue engineering, and composite materials. C. aspersum secretes three mucus-one shielding the animal from environmental threats, one adhesive mucus from the pedal surface of the foot, and another pedal mucus that is lubricating. It remains a mystery how compositional differences account for their substantially different properties. Here, we characterize mucus proteins, glycosylation, ion content, and mechanical properties that could be used to provide insight into structure-function relationships through an integrative "mucomics" approach. We identify macromolecular components of these hydrogels, including a previously unreported protein class termed Conserved Anterior Mollusk Proteins (CAMPs). Revealing differences between C. aspersum mucus shows how considering structure at all levels can inform the design of mucus-inspired materials.

Peculiar Phosphonate Modifications of Velvet Worm Slime Revealed by Advanced Nuclear Magnetic Resonance and Mass Spectrometry.

Nature is rich with examples of highly specialized biological materials produced by organisms for functions, including defense, hunting, and protection. Along these lines, velvet worms (Onychophora) expel a protein-based slime used for hunting and defense that upon shearing and dehydration forms fibers as stiff as thermoplastics. These fibers can dissolve back into their precursor proteins in water, after which they can be drawn into new fibers, providing biological inspiration to design recyclable materials. Elevated phosphorus content in velvet worm slime was previously observed and putatively ascribed to protein phosphorylation. Here, we show instead that phosphorus is primarily present as phosphonate moieties in the slime of distantly related velvet worm species. Using high-resolution nuclear magnetic resonance (NMR), natural abundance dynamic nuclear polarization (DNP), and mass spectrometry (MS), we demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with glycans linked to large slime proteins, while transcriptomic analyses confirm the expression of 2-AEP synthesizing enzymes in slime glands. The evolutionary conservation of this rare protein modification suggests an essential functional role of phosphonates in velvet worm slime and should stimulate further study of the function of this unusual chemical modification in nature.

Characterization of the need for galactofuranose during the Neurospora crassa life cycle.

Galactofuranose is a constituent of the cell walls of filamentous fungi. The galactofuranose can be found as a component of N-linked oligosaccharides, in O-linked oligosaccharides, in GPI-anchored galactomannan, and in free galactomannan. The Neurospora genome contains a single UDP-galactose mutase gene (ugm-1/NCU01824) and two UDP-galactofuranose translocases used to import UDP-galactofuranose into the lumen of the Golgi apparatus (ugt-1/NCU01826 and ugt-2/NCU01456). Our results demonstrate that loss of galactofuranose synthesis or its translocation into the lumen of the secretory pathway affects the morphology and growth rate of the vegetative hyphae, the production of conidia (asexual spores), and dramatically affects the sexual stages of the life cycle. In mutants that are unable to make galactofuranose or transport it into the lumen of the Golgi apparatus, ascospore development is aborted soon after fertilization and perithecium maturation is aborted prior to the formation of the neck and ostiole. The Neurospora genome contains three genes encoding possible galactofuranosyltransferases from the GT31 family of glycosyltransferases (gfs-1/NCU05878, gfs-2/NCU07762, and gfs-3/NCU02213) which might be involved in generating galactofuranose-containing oligosaccharide structures. Analysis of triple KO mutants in GT31 glycosyltransferases shows that these mutants have normal morphology, suggesting that these genes do not encode vital galactofuranosyltransferases.