Viruses direct carbon cycling in lake sediments under global change.

Global change is altering the vast amount of carbon cycled by microbes between land and freshwater, but how viruses mediate this process is poorly understood. Here, we show that viruses direct carbon cycling in lake sediments, and these impacts intensify with future changes in water clarity and terrestrial organic matter (tOM) inputs. Using experimental tOM gradients within sediments of a clear and a dark boreal lake, we identified 156 viral operational taxonomic units (vOTUs), of which 21% strongly increased with abundances of key bacteria and archaea, identified via metagenome-assembled genomes (MAGs). MAGs included the most abundant prokaryotes, which were themselves associated with dissolved organic matter (DOM) composition and greenhouse gas (GHG) concentrations. Increased abundances of virus-like particles were separately associated with reduced bacterial metabolism and with shifts in DOM toward amino sugars, likely released by cell lysis rather than higher molecular mass compounds accumulating from reduced tOM degradation. An additional 9.6% of vOTUs harbored auxiliary metabolic genes associated with DOM and GHGs. Taken together, these different effects on host dynamics and metabolism can explain why abundances of vOTUs rather than MAGs were better overall predictors of carbon cycling. Future increases in tOM quantity, but not quality, will change viral composition and function with consequences for DOM pools. Given their importance, viruses must now be explicitly considered in efforts to understand and predict the freshwater carbon cycle and its future under global environmental change.

LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

Canopy and understory nitrogen additions differently affect soil microbial residual carbon in a temperate forest.

Atmospheric nitrogen (N) deposition in forests can affect soil microbial growth and turnover directly through increasing N availability and indirectly through altering plant-derived carbon (C) availability for microbes. This impacts microbial residues (i.e., amino sugars), a major component of soil organic carbon (SOC). Previous studies in forests have so far focused on the impact of understory N addition on microbes and microbial residues, but the effect of N deposition through plant canopy, the major pathway of N deposition in nature, has not been explicitly explored. In this study, we investigated whether and how the quantities (25 and 50 kg N ha-1 year-1) and modes (canopy and understory) of N addition affect soil microbial residues in a temperate broadleaf forest under 10-year N additions. Our results showed that N addition enhanced the concentrations of soil amino sugars and microbial residual C (MRC) but not their relative contributions to SOC, and this effect on amino sugars and MRC was closely related to the quantities and modes of N addition. In the topsoil, high-N addition significantly increased the concentrations of amino sugars and MRC, regardless of the N addition mode. In the subsoil, only canopy N addition positively affected amino sugars and MRC, implying that the indirect pathway via plants plays a more important role. Neither canopy nor understory N addition significantly affected soil microbial biomass (as represented by phospholipid fatty acids), community composition and activity, suggesting that enhanced microbial residues under N deposition likely stem from increased microbial turnover. These findings indicate that understory N addition may underestimate the impact of N deposition on microbial residues and SOC, highlighting that the processes of canopy N uptake and plant-derived C availability to microbes should be taken into consideration when predicting the impact of N deposition on the C sequestration in temperate forests.

[Non-targeted metabolomics of spleen and liver metabolism in mice treated with Pruni Semen processed with different methods].

Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to investigate the impacts of Pruni Semen processed with different methods(raw and fried) on the liver and spleen metabolism in mice. A total of 24 male mice were randomly assigned to three groups: raw Pruni Semen group, fried Pruni Semen group, and control(deionized water) group. Mice in the three groups were orally administrated with 0.01 g·mL~(-1) Pruni Semen decoction or deionized water for one week. After that, the liver and spleen tissues were collected, and liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis was carried out to investigate the impact of Pruni Semen on the liver and spleen metabolism in mice. Compared with thte control group, the raw Pruni Semen group showed up-regulation of 11 metabolites and down-regulation of 57 metabolites in the spleen(P<0.05), as well as up-regulation of 15 metabolites and down-regulation of 58 metabolites in the liver(P<0.05). The fried Pruni Semen group showed up-regulation of 31 metabolites and down-regulation of 10 metabolites in the spleen(P<0.05), along with up-regulation of 26 metabolites and down-regulation of 61 metabolites in the liver(P<0.05). The differential metabolites identified in the raw Pruni Semen group were primarily associated with alanine, aspartate, and glutamate metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, and D-glutamine and D-glutamate metabolism. The differential metabolites identified in the fried Pruni Semen group predominantly involved riboflavin metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, alanine, aspartate, and glutamate metabolism, D-glutamine and D-glutamate metabolism, and glutathione metabolism. The findings suggest that both raw and fried Pruni Semen have the potential to modulate the metabolism of the liver and spleen in mice by influencing the glutamine and glutamate metabolism.

Systems-wide analysis of the ROK-family regulatory gene rokL6 and its role in the control of glucosamine toxicity in Streptomyces coelicolor.

IMPORTANCE: Central metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate or glucosamine 6-phosphate is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity." We made use of this concept by selecting for suppressors in genes related to glucosamine toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and major facilitator superfamily transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents the toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 shed new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis.

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites.

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galß(1,4)-GlcNAcß(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of ß1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galß(1,4)-GlcNAcß(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

Comprehensive analysis of transcriptome and metabolome analysis reveal new targets of Glaesserella parasuis glucose-specific enzyme IIBC (PtsG).

The ptsG (hpIIBCGlc) gene, belonging to the glucose-specific phosphotransferase system, encodes the bacterial glucose-specific enzyme IIBC. In this study, the effects of a deletion of the ptsG gene were investigated by metabolome and transcriptome analyses. At the transcriptional level, we identified 970 differentially expressed genes between ΔptsG and sc1401 (Padj<0.05) and 2072 co-expressed genes. Among these genes, those involved in methane metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, pyruvate metabolism, phosphotransferase system (PTS), biotin metabolism, Two-component system and Terpenoid backbone biosynthesis showed significant changes in the ΔptsG mutant strain. Metabolome analysis revealed that a total of 310 metabolites were identified, including 20 different metabolites (p < 0.05). Among them, 15 metabolites were upregulated and 5 were downregulated in ΔptsG mutant strain. Statistical analysis revealed there were 115 individual metabolites having correlation, of which 89 were positive and 26 negative. These metabolites include amino acids, phosphates, amines, esters, nucleotides, benzoic acid and adenosine, among which amino acids and phosphate metabolites dominate. However, not all of these changes were attributable to changes in mRNA levels and must also be caused by post-transcriptional regulatory processes. The knowledge gained from this lays the foundation for further study on the role of ptsG in the pathogenic process of Glaesserella parasuis (G.parasuis).

Characterization of the Aminosugar Biosynthetic and Regulatory Genes of Vicenistatin in Monodonata labio-Associated Streptomyces parvus SCSIO Mla-L010.

Vicenistatin (1) is a potent polyketide antitumor antibiotic composed of a 20-membered macrolactam core appended to a unique aminosugar, vicenisamine. In this study, vicenistatin was isolated and its biosynthetic gene cluster identified from Monodonata labio-associated Streptomyces parvus SCSIO Mla-L010. A set of five genes, vicC, vicD, vicE, vicF, and vicG, was confirmed to be involved in the biosynthesis of the aminosugar by gene inactivations. VicG was characterized as an N-methyltransferase that catalyzes the methylation of the 4'-amino group in the last step of the aminosugar biosynthetic pathway; the N-demethyl intermediate 4'-N-demethylvicenistatin (2) was isolated from the ΔvicG mutant strain. In addition, vicR1 was characterized as a positive pathway-specific regulatory gene. Notably, N-demethyl compound 2 was found to exert impressive antibacterial activities, with MIC values spanning 0.06-4 µg/mL, against a panel of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus, Gram-negative Helicobacter pylori, and mycobacterium Mycobacterium smegmatis and the fungal pathogen Candida albicans. Compound 2 was also found to display reduced cytotoxicities relative to vicenistatin, especially against noncancerous human cell lines.

Optimization of environmental factors in a dual in vitro biofilm model of Candida albicans-Streptococcus mutans.

The biofilm formation of Streptococcus mutans-Candida albicans is an important virulence factor for dental caries. The purpose of this study was to determine the effect of some environmental conditions on the biofilm formation like inoculation concentration, temperature, sugar, amino acid, metal ions and saliva, and then establish a persistent in vitro biofilm model for further research. Based on the single factor experiment, the factors participating in the biofilm formation including sugar, inoculation concentration, and saliva increased the biofilm mass, while amino acid, metal ions, temperatures reduced biofilm mass. Optimal conditions for biofilm formation were the inoculation dosage of S. mutans and C. albicans of 108 and 107 , respectively, the addition of 0·3 g l-1 sucrose and sterile saliva. These results contribute to a deep understanding of the factors involved in oral biofilm formation of the important cariogenic pathogen S. mutans and the opportunistic pathogen C. albicans to study better for biofilm and promote the design of new therapeutic approaches. The present research also provides a model for evaluating the therapeutic potential for drugs in the future.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

Acute toxic effects of diclofenac exposure on freshwater crayfish (Procambarus clarkii): Insights from hepatopancreatic pathology, molecular regulation and intestinal microbiota.

In this study, we exposed adult male crayfish (Procambarus clarkii) to different concentrations of diclofenac (DCF) for 96 h. In the meantime, we investigated the alternations of hepatopancreatic pathology, molecular regulation and intestinal microbiota of P. clarkii exposed to DCF. The results demonstrated DCF led to histological changes including epithelium vacuolization and tubule lumen dilatation in the hepatopancreas. Transcriptome sequencing analysis showed that 642 and 586 genes were differentially expressed in the hepatopancreas of P. clarkii exposed to 1 and 10 mg/L DCF, respectively. DCF could affect the functions of antioxidation, immunity and metabolism of hepatopancreas by inducing the abnormal expressions of immune- and redox-related genes. GO enrichment results demonstrated that 10 mg/L DCF exposure could modulate the processes of molting, amino sugar metabolism, protein hydrolysis and intracellular protein translocation of P. clarkii. Additionally, the abundances of bacterial families including Shewanellaceae, Bacteroidaceae, Vibrionaceae, Erysipelotrichaceae, Aeromonadaceae, Moraxellaceae, etc. in the intestine were significantly changed after DCF exposure, and the disruption of intestinal flora might further cause abnormal intestinal metabolism in P. clarkii. This study provides novel mechanistic insights into the toxic effects of anti-inflammatory drugs on aquatic crustaceans.

Regulation of bacterial metabolism by small RNAs using diverse mechanisms.

Bacteria live in many dynamic environments with alternating cycles of feast or famine that have resulted in the evolution of mechanisms to quickly alter their metabolic capabilities. Such alterations often involve complex regulatory networks that modulate expression of genes involved in nutrient uptake and metabolism. A great number of protein regulators of metabolism have been characterized in depth. However, our ever-increasing understanding of the roles played by RNA regulators has revealed far greater complexity to regulation of metabolism in bacteria. Here, we review the mechanisms and functions of selected bacterial RNA regulators and discuss their importance in modulating nutrient uptake as well as primary and secondary metabolic pathways.

Deciphering the fate of slan+ -monocytes in human tonsils by gene expression profiling.

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan+ -monocytes. In recent studies, we and other groups have reported that, in tonsils, slan marks dendritic cell (DC)-like cells, as defined by morphological, phenotypical, and functional criteria. However, subsequent investigations in lymphomas have uncovered a significant heterogeneity of tumor-infiltrating slan+ -cells, including a macrophage-like state. Based on their emerging role in tissue inflammation and cancer, herein we investigated slan+ -cell fate in tonsils by using a molecular-based approach. Hence, RNA from tonsil slan+ -cells, conventional CD1c+ DCs (cDC2) and CD11b+ CD14+ -macrophages was subjected to gene expression analysis. For comparison, transcriptomes were also obtained from blood cDC2, classical (CL), intermediate (INT), NC, and slan+ -monocytes. Data demonstrate that the main trajectory of human slan+ -monocytes infiltrating the tonsil tissue is toward a macrophage-like population, displaying molecular features distinct from those of tonsil CD11b+ CD14+ -macrophages and cDC2. These findings provide a novel view on the terminal differentiation path of slan+ -monocytes, which is relevant for inflammatory diseases and lymphomas.

Recombinant SARS-CoV-2 S Protein Binds to Glycans of the Lactosamine Family in vitro.

Many viruses, beside binding to their main cell target, interact with other molecules that promote virus adhesion to the cell; often, these additional targets are glycans. The main receptor for SARS-CoV-2 is a peptide motif in the ACE2 protein. We studied interaction of the recombinant SARS-CoV-2 spike (S) protein with an array of glycoconjugates, including various sialylated, sulfated, and other glycans, and found that the S protein binds some (but not all) glycans of the lactosamine family. We suggest that parallel influenza infection will promote SARS-CoV-2 adhesion to the respiratory epithelial cells due to the unmasking of lactosamine chains by the influenza virus neuraminidase.

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN, or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each species of bacteria when it was cultured alone. Likewise, cocultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different from the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernatants. Differing from what was found in a previous report, growth of S. mutans alone with GlcN inhibited the expression of multiple operons required for mutacin production. Cocultivation with S. gordonii consistently increased the expression of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes in S. mutans Conversely, S. gordonii appeared to be less affected by the presence of S. mutans but did show increases in genes for biosynthetic processes in the cocultures. In conclusion, amino sugars profoundly alter the interactions between pathogenic and commensal streptococci by reprogramming central metabolism.IMPORTANCE Carbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii), in an RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose.

BACKGROUND: The exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains. RESULTS: In this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant-) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA. CONCLUSIONS: Screening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

The BRAF inhibitor PLX4032 is effective in treating BRAF-mutated melanoma; however, because drug resistance develops in most cases, it is critical to develop a new strategy for inhibiting drug-resistant melanoma growth. The melanoma-associated membrane glycoprotein CD63 is involved in cell proliferation and metastasis. Here, we found that cell surface CD63 suppresses the proliferation of human melanoma cells and PLX4032-resistant cells. Endogenous CD63 protein levels were negatively correlated with PLX4032 resistance of human melanoma cell lines. CD63 overexpression in these cells, in which endogenous CD63 levels are low, suppressed cell proliferation under PLX4032 treatment. The cell surface levels and average molecular mass of CD63 were increased with PLX4032 treatment because of the up-regulated polylactosamine modification caused by induced ß1,3- N-acetylglucosaminyltransferase 2 expression, which is involved in polylactosamine synthesis. Forced cell surface localization of CD63 led to reduced melanoma cell proliferation without PLX4032 treatment. CD63 overexpression in PLX4032-resistant cells, in which CD63 levels were lower and cell surface polylactosamine levels were higher than those in parental cells, effectively suppressed proliferation. Our study shows the potential of CD63 to sensitize melanoma cells to PLX4032 and to reduce the proliferation of PLX4032-resistant cells.-Kudo, K., Yoneda, A., Sakiyama, D., Kojima, K., Miyaji, T., Yamazaki, M., Yaita, S., Hyodo, T., Satow, R., Fukami, K. Cell surface CD63 increased by up-regulated polylactosamine modification sensitizes human melanoma cells to the BRAF inhibitor PLX4032.

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

T-cell activation by transitory neo-antigens derived from distinct microbial pathways.

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.