Functional genomics identifies N-acetyllactosamine extension of complex N-glycans as a mechanism to evade lysis by natural killer cells.

Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.

[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.

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.

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).

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.

Differential Timing for Glucose Assimilation in Prochlorococcus and Coexistent Microbial Populations in the North Pacific Subtropical Gyre.

The marine cyanobacterium Prochlorococcus can utilize glucose as a source of carbon. However, the relative importance of inorganic and organic carbon assimilation and the timing of glucose assimilation are still poorly understood in these numerically dominant cyanobacteria. Here, we investigated whole microbial community and group-specific primary production and glucose assimilation using incubations with radioisotopes combined with flow cytometry cell sorting. We also studied changes in the microbial community structure in response to glucose enrichments and analyzed the transcription of Prochlorocccus genes involved in carbon metabolism and photosynthesis. Our results showed a diel variation for glucose assimilation in Prochlorococcus, with maximum assimilation at midday and minimum at midnight (~2-fold change), which was different from that of the total microbial community. This suggests that the timing in glucose assimilation in Prochlorococcus is coupled to photosynthetic light reactions producing energy, it being more convenient for Prochlorococcus to show maximum glucose uptake precisely when the rest of microbial populations have their minimum glucose uptake. Many transcriptional responses to glucose enrichment occurred after 12- and 24-h periods, but community composition did not change. High-light Prochlorococcus strains were the most impacted by glucose addition, with transcript-level increases observed for genes in pathways for glucose metabolism, such as the pentose phosphate pathway, the Entner-Doudoroff pathway, glycolysis, respiration, and glucose transport. While Prochlorococcus C assimilation from glucose represented less than 0.1% of the bacterium's photosynthetic C fixation, increased assimilation during the day and glcH gene upregulation upon glucose enrichment indicate an important role of mixotrophic C assimilation by natural populations of Prochlorococcus. IMPORTANCE Several studies have demonstrated that Prochlorococcus, the most abundant photosynthetic organism on Earth, can assimilate organic molecules, such as amino acids, amino sugars, ATP, phosphonates, and dimethylsulfoniopropionate. This autotroph can also assimilate small amounts of glucose, supporting the hypothesis that Prochlorococcus is mixotrophic. Our results show, for the first time, a diel variability in glucose assimilation by natural populations of Prochlorococcus with maximum assimilation during midday. Based on our previous results, this indicates that Prochlorococcus could maximize glucose uptake by using ATP made during the light reactions of photosynthesis. Furthermore, Prochlorococcus showed a different timing of glucose assimilation from the total population, which may offer considerable fitness advantages over competitors "temporal niches." Finally, we observed transcriptional changes in some of the genes involved in carbon metabolism, suggesting that Prochlorococcus can use both pathways previously proposed in cyanobacteria to metabolize glucose.

Mechanism exploration of Solanum nigrum L. hyperaccumulating Cd compared to Zn from the perspective of metabolic pathways based on differentially expressed proteins using iTRAQ.

It is challenging to determine the mechanism involved in only Cd hyperaccumulation by Solanum nigrum L. owing to the uniqueness of the process. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to explore the mechanism by which S. nigrum hyperaccumulates Cd by comparing the differentially expressed proteins (DEPs) for Cd and Zn accumulation (non-Zn hyperaccumulator). Based on the comparison between the DEPs associated with Cd and Zn accumulation, the relative metabolic pathways reflected by 17 co-intersecting specific proteins associated with Cd and Zn accumulation included phagosome, aminoacyl-tRNA biosynthesis, and carbon metabolism. Apart from the 17 co-intersecting specific proteins, the conjoint metabolic pathways reported by 21 co-intersecting specific proteins associated with Cd accumulation and 30 co-intersecting specific proteins associated with Zn accumulation, the most differentially expressed metabolic pathways might cause Cd TF (Translocation factor)> 1 and Zn TF< 1, including protein export, ribosome, amino sugar, and nucleotide sugar metabolism. The determined DEPs were verified using qRT-PCR with the four key proteins M1CW30, A0A3Q7H652, A0A0V0IFB9, and A0A0V0IAC4. The plasma membrane H+-ATPase protein was identified using western blotting. Some physiological indices for protein-related differences indirectly confirmed the above results. These results are crucial to further explore the mechanisms involved in Cd hyperaccumulation.

Gut Microbiota Protected Against pseudomonas aeruginosa Pneumonia via Restoring Treg/Th17 Balance and Metabolism.

Backgrounds and Purpose: The theory of "entero-pulmonary axis" proves that pneumonia leads to gut microbiota disturbance and Treg/Th17 immune imbalance. This study is aimed to explore the potential mechanism of fecal microbiota transplantation (FMT) in the treatment of Pseudomonas aeruginosa pneumonia, in order to provide new insights into the treatment of pneumonia. Methods: Pseudomonas aeruginosa and C57/BL6 mice were used to construct the acute pneumonia mouse model, and FMT was treated. Histopathological changes in lung and spleen were observed by HE staining. The expression of CD25, Foxp3 and IL-17 was observed by immunofluorescence. The proportion of Treg and Th17 cells was analyzed by flow cytometry. Serum IL-6, LPS, and IFN-γ levels were detected by ELISA. The expression of TNF-α, IFN-γ, IL-6, IL-2, Foxp3, IL-17, IL-10, and TGFß1 in lung tissue homogenate was detected by qRT-PCR. 16S rRNA sequencing and non-targeted metabolomics were used to analyze gut microbiota and metabolism. Results: Pseudomonas aeruginosa caused the decrease of body weight, food and water intake, lung tissue, and spleen injury in mice with pneumonia. Meanwhile, it caused lung tissue and serum inflammation, and Treg/Th17 cell imbalance in mice with pneumonia. Pseudomonas aeruginosa reduced the diversity and number of gut microbiota in pneumonia mice, resulting in metabolic disorders, superpathway of quinolone and alkylquinolone biosynthesis. It also led to the decrease of 2-heptyl-3-hydroxy-4(1H)-quinolone biosynthesis, and the enrichment of Amino sugar and nucleotide sugar metabolism. FMT with or without antibiotic intervention restored gut microbiota abundance and diversity, suppressed inflammation and tissue damage, and promoted an immunological balance of Treg/Th17 cells in mice with pneumonia. In addition, FMT inhibited the aerobactin biosynthesis, 4-hydroxyphenylacetate degradation, superpathway of lipopolysaccharide biosynthesis and L-arabinose degradation IV function of microbiota, and improved amino sugar and nucleotide sugar metabolism. Conclusions: FMT restored the Treg/Th17 cells' balance and improved inflammation and lung injury in mice with Pseudomonas aeruginosa pneumonia by regulating gut microbiota disturbance and metabolic disorder.

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.

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.

Modulation of the carbohydrate-binding specificity of two Xenopus proto-type galectins by site-directed mutagenesis.

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 ß-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.

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.

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.

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.

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.

Exploring Amino Sugar and Phosphoenolpyruvate Metabolism to Improve Escherichia coli N-Acetylneuraminic Acid Production.

N-acetyl-d-neuraminic acid (NeuAc) has attracted considerable attention because of its wide-ranging applications. The use of cheap carbon sources such as glucose without the addition of any precursor in microbial NeuAc production has many advantages. In this study, improved NeuAc production was attained through the optimization of amino sugar metabolism pathway kinetics and reservation of a phosphoenolpyruvate (PEP) pool in Escherichia coli. N-acylglucosamine 2-epimerase and N-acetylneuraminate synthase from different sources and their best combinations were used to obtain optimized enzyme kinetics and expression intensity, which resulted in a significant increase in NeuAc production. Next, after a design was engineered for enabling the PEP metabolic pathway to retain the PEP pool, the production of NeuAc reached 16.7 g/L, which is the highest NeuAc production rate that has been reported from using glucose as the sole carbon source.

The Sialoside-Binding Pocket of SARS-CoV-2 Spike Glycoprotein Structurally Resembles MERS-CoV.

COVID-19 novel coronavirus (CoV) disease caused by severe acquired respiratory syndrome (SARS)-CoV-2 manifests severe lethal respiratory illness in humans and has recently developed into a worldwide pandemic. The lack of effective treatment strategy and vaccines against the SARS-CoV-2 poses a threat to human health. An extremely high infection rate and multi-organ secondary infection within a short period of time makes this virus more deadly and challenging for therapeutic interventions. Despite high sequence similarity and utilization of common host-cell receptor, human angiotensin-converting enzyme-2 (ACE2) for virus entry, SARS-CoV-2 is much more infectious than SARS-CoV. Structure-based sequence comparison of the N-terminal domain (NTD) of the spike protein of Middle East respiratory syndrome (MERS)-CoV, SARS-CoV, and SARS-CoV-2 illustrate three divergent loop regions in SARS-CoV-2, which is reminiscent of MERS-CoV sialoside binding pockets. Comparative binding analysis with host sialosides revealed conformational flexibility of SARS-CoV-2 divergent loop regions to accommodate diverse glycan-rich sialosides. These key differences with SARS-CoV and similarity with MERS-CoV suggest an evolutionary adaptation of SARS-CoV-2 spike glycoprotein reciprocal interaction with host surface sialosides to infect host cells with wide tissue tropism.

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.

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.