-
PLoS Pathogens Jun 2024The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite....
The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.
PubMed: 38900808
DOI: 10.1371/journal.ppat.1011979 -
International Journal of Molecular... Jun 2024Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases....
Diabetic retinopathy (DR) is a very serious diabetes complication. Changes in the O-linked N-acetylglucosamine (O-GlcNAc) modification are associated with many diseases. However, its role in DR is not fully understood. In this research, we explored the effect of O-GlcNAc modification regulation by activating AMP-activated protein kinase (AMPK) in DR, providing some evidence for clinical DR treatment in the future. Bioinformatics was used to make predictions from the database, which were validated using the serum samples of diabetic patients. As an in vivo model, diabetic mice were induced using streptozotocin (STZ) injection with/without an AMPK agonist (metformin) or an AMPK inhibitor (compound C) treatment. Electroretinogram (ERG) and H&E staining were used to evaluate the retinal functional and morphological changes. In vitro, 661 w cells were exposed to high-glucose conditions, with or without metformin treatment. Apoptosis was evaluated using TUNEL staining. The protein expression was detected using Western blot and immunofluorescence staining. The angiogenesis ability was detected using a tube formation assay. The levels of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in the serum changed in the DR patients in the clinic. In the diabetic mice, the ERG wave amplitude and retinal thickness decreased. In vitro, the apoptotic cell percentage and Bax expression were increased, and Bcl2 expression was decreased in the 661 w cells under high-glucose conditions. The O-GlcNAc modification was increased in DR. In addition, the expression of GFAT/TXNIP O-GlcNAc was also increased in the 661 w cells after the high-glucose treatment. Additionally, the Co-immunoprecipitation(CO-IP) results show that TXNIP interacted with the O-GlcNAc modification. However, AMPK activation ameliorated this effect. We also found that silencing the AMPKα1 subunit reversed this process. In addition, the conditioned medium of the 661 w cells may have affected the tube formation in vitro. Taken together, O-GlcNAc modification was increased in DR with photoreceptor cell degeneration and neovascularization; however, it was reversed after activating AMPK. The underlying mechanism is linked to the GFAT/TXNIP-O-GlcNAc modification signaling axis. Therefore, the AMPKα1 subunit plays a vital role in the process.
Topics: Diabetic Retinopathy; Animals; Mice; Acetylglucosamine; N-Acetylglucosaminyltransferases; Diabetes Mellitus, Experimental; Humans; AMP-Activated Protein Kinases; Male; Apoptosis; Metformin; beta-N-Acetylhexosaminidases; Retina; Mice, Inbred C57BL; Cell Line
PubMed: 38892474
DOI: 10.3390/ijms25116286 -
Acta Biochimica Et Biophysica Sinica Jun 2024deploys various morphological forms through complex switching mechanisms, ensuring its survival and thriving as a commensal or pathogen in vastly different human...
deploys various morphological forms through complex switching mechanisms, ensuring its survival and thriving as a commensal or pathogen in vastly different human niches. In this study, we demonstrate that a novel ''rod'' morphological form of . coexists and is interchangeable with previously reported white, gray, and opaque forms, constituting a tetra-stable phenotypic switching system. Rod cells arise from the mutant of SC5314 cells or from the clinical BJ1097 strain cultured under glucose-free conditions. They are characterized by a distinct gene expression profile and can be stably maintained through passaging or inhabitation of the gastrointestinal (GI) tract of mice. Remarkably, the majority of the mutant cells become rod cells in N-acetylglucosamine (GlcNAc)-containing medium, and the GlcNAc sensor Ngs1 is instrumental in converting the white or gray cells to the rod cells. Conversely, glucose inhibits rod cells through Cph1; consequently, the loss of Cph1 in the mutantcells permits their conversion to rod cells in glucose-replete media. Notably, rod cells of the / mutant display superior adaptation and longer persistence in the murine GI environment than wild-type white cells. Taken together, these findings establish rod cells as a previously unappreciated form that is not only morphologically and transcriptionally distinguishable but also defined by specific genetic and environmental determinants, shedding light on complex fungus-host interactions.
PubMed: 38887798
DOI: 10.3724/abbs.2024066 -
ELife Jun 2024Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and...
Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.
Topics: Chitinases; Animals; Hydrogen-Ion Concentration; Mice; Molecular Dynamics Simulation; Chitin; Protein Conformation; Crystallography, X-Ray; Protein Binding; Ligands; Kinetics; Acetylglucosamine; Models, Molecular
PubMed: 38884443
DOI: 10.7554/eLife.89918 -
Pesticide Biochemistry and Physiology Jun 2024Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely...
Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely elucidated, nor has its disturbing effect on chitin synthesis genes. In this study, bioassay results demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that five chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increase in larval susceptibilities to LC lufenuron by 75.48%, 65.00%, 68.42% and 28.00%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and proved that the combination of RNAi and lufenuron would improve the control effect of this pest.
Topics: Animals; Chitin; Benzamides; Larva; Insecticides; Moths; Insect Proteins; Chitin Synthase; Helicoverpa armigera; Fluorocarbons
PubMed: 38879310
DOI: 10.1016/j.pestbp.2024.105962 -
Applied Microbiology and Biotechnology Jun 2024The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a...
The lincoamide antibiotic lincomycin, derived from Streptomyces lincolnensis, is widely used for the treatment of infections caused by gram-positive bacteria. As a common global regulatory factor of GntR family, DasR usually exists as a regulatory factor that negatively regulates antibiotic synthesis in Streptomyces. However, the regulatory effect of DasR on lincomycin biosynthesis in S. lincolnensis has not been thoroughly investigated. The present study demonstrates that DasR functions as a positive regulator of lincomycin biosynthesis in S. lincolnensis, and its overexpression strain OdasR exhibits a remarkable 7.97-fold increase in lincomycin production compared to the wild-type strain. The effects of DasR overexpression could be attenuated by the addition of GlcNAc in the medium in S. lincolnensis. Combined with transcriptome sequencing and RT-qPCR results, it was found that most structural genes in GlcNAc metabolism and central carbon metabolism were up-regulated, but the lincomycin biosynthetic gene cluster (lmb) were down-regulated after dasR knock-out. However, DasR binding were detected with the DasR responsive elements (dre) of genes involved in GlcNAc metabolism pathway through electrophoretic mobility shift assay, while they were not observed in the lmb. These findings will provide novel insights for the genetic manipulation of S. lincolnensis to enhance lincomycin production. KEY POINTS: • DasR is a positive regulator that promotes lincomycin synthesis and does not affect spore production • DasR promotes lincomycin production through indirect regulation • DasR correlates with nutrient perception in S. lincolnensis.
Topics: Lincomycin; Streptomyces; Gene Expression Regulation, Bacterial; Anti-Bacterial Agents; Bacterial Proteins; Multigene Family; Acetylglucosamine; Biosynthetic Pathways; Gene Expression Profiling
PubMed: 38878095
DOI: 10.1007/s00253-024-13201-7 -
GeroScience Jun 2024Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly...
Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age.For precision analysis, three different plasma pools were analysed over 26 days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3 months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10 years, respectively. Samples were collected every 3 months and 3 weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits.The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits.The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths.
PubMed: 38877341
DOI: 10.1007/s11357-024-01239-4 -
Computers in Biology and Medicine Jun 2024Neisseria meningitidis, commonly known as the meningococcus, leads to substantial illness and death among children and young adults globally, revealing as either...
An integrated in silico approach for the identification of novel potential drug target and chimeric vaccine against Neisseria meningitides strain 331401 serogroup X by subtractive genomics and reverse vaccinology.
Neisseria meningitidis, commonly known as the meningococcus, leads to substantial illness and death among children and young adults globally, revealing as either epidemic or sporadic meningitis and/or septicemia. In this study, we have designed a novel peptide-based chimeric vaccine candidate against the N. meningitidis strain 331,401 serogroup X. Through rigorous analysis of subtractive genomics, two essential cytoplasmic proteins, namely UPI000012E8E0(UDP-3-O-acyl-GlcNAc deacetylase) and UPI0000ECF4A9(UDP-N-acetylglucosamine acyltransferase) emerged as potential drug targets. Additionally, using reverse vaccinology, the outer membrane protein UPI0001F4D537 (Membrane fusion protein MtrC) identified by subcellular localization and recognized for its known indispensable role in bacterial survival was identified as a novel chimeric vaccine target. Following a careful comparison of MHC-I, MHC-II, T-cell, and B-cell epitopes, three epitopes derived from UPI0001F4D537 were linked with three types of linkers-GGGS, EAAAK, and the essential PADRE-for vaccine construction. This resulted in eight distinct vaccine models (V1-V8). Among them V1 model was selected as the final vaccine construct. It exhibits exceptional immunogenicity, safety, and enhanced antigenicity, with 97.7 % of its residues in the Ramachandran plot's most favored region. Subsequently, the vaccine structure was docked with the TLR4/MD2 complex and six different HLA allele receptors using the HADDOCK server. The docking resulted in the lowest HADDOCK score of 39.3 ± 9.0 for TLR/MD2. Immune stimulation showed a strong immune response, including antibodies creation and the activation of B-cells, T Cytotoxic cells, T Helper cells, Natural Killer cells, and interleukins. Furthermore, the vaccine construct was successfully expressed in the Escherichia coli system by reverse transcription, optimization, and ligation in the pET-28a (+) vector for the expression study. The current study proposes V1 construct has the potential to elicit both cellular and humoral responses, crucial for the developing an epitope-based vaccine against N. meningitidis strain 331,401 serogroup X.
PubMed: 38870724
DOI: 10.1016/j.compbiomed.2024.108738 -
Synthetic and Systems Biotechnology Dec 2024Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial...
Amino oligosaccharides (AOs) possess various biological activities and are valuable in the pharmaceutical, food industries, and agriculture. However, the industrial manufacturing of AOs has not been realized yet, despite reports on physical, chemical, and biological approaches. In this study, the production of chitin oligosaccharides (CHOS), a type of structurally defined AOs, was achieved in through combinatorial pathway engineering. The most suitable glycosyltransferase for CHOS production was found to be NodCL from . Then, by knocking out the gene to block the flow of N-acetyl-d-glucosamine (NAG) to the glycolytic pathway in and adjusting the copy number of NodCL-coding gene, the CHOS yield was increased by 6.56 times. Subsequently, by introducing of UDP-N-acetylglucosamine (UDP-GlcNAc) pathway for and optimizing fermentation conditions, the yield of CHOS reached 207.1 and 468.6 mg/L in shake-flask cultivation and a 5-L fed-batch bioreactor, respectively. Meanwhile, the concentration of UDP-GlcNAc was 91.0 mg/L, the highest level reported in so far. This study demonstrated, for the first time, the production of CHOS with distinct structures in plasmid-free , laying the groundwork for the biosynthesis of CHOS and providing a starting point for further engineering and commercial production.
PubMed: 38868610
DOI: 10.1016/j.synbio.2024.05.011 -
Journal of Agricultural and Food... Jun 2024The addition of the -linked -acetylglucosamine (-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products....
The addition of the -linked -acetylglucosamine (-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products. However, the synthesis of terpenoid glycoside derivatives decorated with GlcNAc remains a challenging task due to the absence of glycosyltransferases, key enzymes for catalyzing the transfer of GlcNAc to terpenoids. In this study, we demonstrated that the enzyme mutant UGT74AC1 efficiently transferred GlcNAc from uridine diphosphate (UDP)-GlcNAc to a variety of terpenoids. This powerful enzyme was employed to synthesize GlcNAc-decorated derivatives of terpenoids, including mogrol, steviol, andrographolide, protopanaxadiol, glycyrrhetinic acid, ursolic acid, and betulinic acid for the first time. To unravel the mechanism of UDP-GlcNAc recognition, we determined the X-ray crystal structure of the inactivated mutant UGT74AC1 in complex with UDP-GlcNAc at a resolution of 1.66 Å. Through molecular dynamic simulation and activity analysis, we revealed the molecular mechanism and catalytically important amino acids directly involved in the recognition of UDP-GlcNAc. Overall, this study not only provided a potent biocatalyst capable of glycodiversifying natural products but also elucidated the structural basis for UDP-GlcNAc recognition by glycosyltransferases.
Topics: Acetylglucosamine; Glycosides; Glycosyltransferases; Terpenes; Plant Proteins; Biocatalysis
PubMed: 38867497
DOI: 10.1021/acs.jafc.4c02832