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BMC Infectious Diseases Jun 2024Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from...
BACKGROUND
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes.
METHODS
Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm).
RESULTS
We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M.S. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and HO. Since schistosomes are damaged by HO, we determined if SmNPP5 could impede HO production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M.S.
CONCLUSIONS
The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.
Topics: Riboflavin; Flavin Mononucleotide; Animals; Flavin-Adenine Dinucleotide; Schistosoma mansoni; Mice; Humans; Schistosomiasis mansoni
PubMed: 38918706
DOI: 10.1186/s12879-024-09538-z -
Nature Communications Jun 2024Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the...
Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.
Topics: Mucopolysaccharidosis III; Humans; Lysosomes; Acetyltransferases; Cryoelectron Microscopy; Catalytic Domain; Mutation; Heparitin Sulfate; Acetyl Coenzyme A; Models, Molecular; Glucosamine; Acetylation; Intracellular Membranes
PubMed: 38918376
DOI: 10.1038/s41467-024-49614-1 -
Journal of Nanobiotechnology Jun 2024Photothermal therapy (PTT) is a promising cancer treatment method due to its ability to induce tumor-specific T cell responses and enhance therapeutic outcomes. However,...
Photothermal therapy (PTT) is a promising cancer treatment method due to its ability to induce tumor-specific T cell responses and enhance therapeutic outcomes. However, incomplete PTT can leave residual tumors that often lead to new metastases and decreased patient survival in clinical scenarios. This is primarily due to the release of ATP, a damage-associated molecular pattern that quickly transforms into the immunosuppressive metabolite adenosine by CD39, prevalent in the tumor microenvironment, thus promoting tumor immune evasion. This study presents a photothermal nanomedicine fabricated by electrostatic adsorption among the Fe-doped polydiaminopyridine (Fe-PDAP), indocyanine green (ICG), and CD39 inhibitor sodium polyoxotungstate (POM-1). The constructed Fe-PDAP@ICG@POM-1 (FIP) can induce tumor PTT and immunogenic cell death when exposed to a near-infrared laser. Significantly, it can inhibit the ATP-adenosine pathway by dual-directional immunometabolic regulation, resulting in increased ATP levels and decreased adenosine synthesis, which ultimately reverses the immunosuppressive microenvironment and increases the susceptibility of immune checkpoint blockade (aPD-1) therapy. With the aid of aPD-1, the dual-directional immunometabolic regulation strategy mediated by FIP can effectively suppress/eradicate primary and distant tumors and evoke long-term solid immunological memory. This study presents an immunometabolic control strategy to offer a salvage option for treating residual tumors following incomplete PTT.
Topics: Animals; Photothermal Therapy; Immunotherapy; Mice; Nanomedicine; Tumor Microenvironment; Cell Line, Tumor; Humans; Indocyanine Green; Neoplasms; Adenosine Triphosphate; Adenosine; Mice, Inbred C57BL; Apyrase; Female; Phototherapy
PubMed: 38915007
DOI: 10.1186/s12951-024-02643-w -
ELife Jun 2024Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range...
Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic subunit of protein kinase A (PKA-C). This long-range synergistic action is involved in substrate recognition and fidelity, and it may also regulate PKA's association with regulatory subunits and other binding partners. To date, a complete understanding of this intramolecular mechanism is still lacking. Here, we integrated NMR(Nuclear Magnetic Resonance)-restrained molecular dynamics simulations and a Markov State Model to characterize the free energy landscape and conformational transitions of PKA-C. We found that the apoenzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the αC-β4 loop. We validated the second excited state by analyzing the F100A mutant of PKA-C, assessing its structural response to ATP and substrate binding. While PKA-C preserves its catalytic efficiency with Kemptide, this mutation rearranges the αC-β4 loop conformation, interrupting the coupling of the two lobes and abolishing the allosteric binding cooperativity. The highly conserved αC-β4 loop emerges as a pivotal element to control the synergistic binding of nucleotide and substrate, explaining how mutations or insertions near or within this motif affect the function and drug sensitivity in homologous kinases.
Topics: Molecular Dynamics Simulation; Allosteric Regulation; Adenosine Triphosphate; Catalytic Domain; Cyclic AMP-Dependent Protein Kinases; Protein Conformation; Protein Binding; Nucleotides; Substrate Specificity; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
PubMed: 38913408
DOI: 10.7554/eLife.91506 -
Virulence Dec 2024β-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout...
β-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent β-lactam resistance in . However, NagZ's role in the virulence of remains unclear. Our study, incorporating data on mouse and larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ's function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.
Topics: Animals; Virulence; Mice; Enterobacter cloacae; Larva; Moths; Acetylglucosaminidase; Cyclic GMP; Enterobacteriaceae Infections; Virulence Factors; Bacterial Proteins; Female; Gene Expression Regulation, Bacterial; Gene Knockout Techniques
PubMed: 38912723
DOI: 10.1080/21505594.2024.2367652 -
Scientific Reports Jun 2024Microglia, brain-resident macrophages, can acquire distinct functional phenotypes, which are supported by differential reprogramming of cell metabolism. These...
Microglia, brain-resident macrophages, can acquire distinct functional phenotypes, which are supported by differential reprogramming of cell metabolism. These adaptations include remodeling in glycolytic and mitochondrial metabolic fluxes, potentially altering energy substrate availability at the tissue level. This phenomenon may be highly relevant in the brain, where metabolism must be precisely regulated to maintain appropriate neuronal excitability and synaptic transmission. Direct evidence that microglia can impact on neuronal energy metabolism has been widely lacking, however. Combining molecular profiling, electrophysiology, oxygen microsensor recordings and mathematical modeling, we investigated microglia-mediated disturbances in brain energetics during neuroinflammation. Our results suggest that proinflammatory microglia showing enhanced nitric oxide release and decreased CX3CR1 expression transiently increase the tissue lactate/glucose ratio that depends on transcriptional reprogramming in microglia, not in neurons. In this condition, neuronal network activity such as gamma oscillations (30-70 Hz) can be fueled by increased ATP production in mitochondria, which is reflected by elevated oxygen consumption. During dysregulated inflammation, high energy demand and low glucose availability can be boundary conditions for neuronal metabolic fitness as revealed by kinetic modeling of single neuron energetics. Collectively, these findings indicate that metabolic flexibility protects neuronal network function against alterations in local substrate availability during moderate neuroinflammation.
Topics: Animals; Neurons; Energy Metabolism; Microglia; Mice; Neuroinflammatory Diseases; Glucose; Mitochondria; Nitric Oxide; Lactic Acid; Nerve Net; Brain; Oxygen Consumption; Adenosine Triphosphate; Inflammation; Male; Mice, Inbred C57BL
PubMed: 38909138
DOI: 10.1038/s41598-024-64872-1 -
Journal of Dairy Science Jun 2024Interventions targeting the gut microbiota, such as fecal microbiota transplantation, prove effective in repairing the intestinal barrier and facilitating the recovery...
Changes in Rumen Epithelial Morphology and Transcriptome, Rumen Metabolome, and Blood Biochemical Parameters in Lactating Dairy Cows with Subacute Rumen Acidosis Following Rumen Content Transplantation.
Interventions targeting the gut microbiota, such as fecal microbiota transplantation, prove effective in repairing the intestinal barrier and facilitating the recovery of its function and metabolism. However, the regulatory mechanisms governing the remodeling of rumen epithelial morphology and function, rumen metabolism, and host metabolism in cows of subacute ruminal acidosis (SARA) remain poorly understood. Here, we explored the changes in rumen epithelial morphology and transcriptome, rumen metabolome, and blood biochemical parameters in SARA cows following rumen content transplantation (RCT). The entire experiment consisted of 2 periods: the SARA induction period and the RCT period. During the SARA induction period, 12 ruminally cannulated lactating Holstein cows were randomly allocated into 2 groups, fed either a conventional diet [CON; n = 4; 40% concentrate, dry matter (DM) basis] or a high-grain diet (HG; n = 8; 60% concentrate, DM basis). Following the SARA induction period, the RCT period started. The HG cows were randomly assigned to 2 groups: the donor-recipient (DR) group and the self-recipient (SR) group. Rumen contents were entirely removed from both groups before RCT. For the DR group, cows were administered 70% rumen content from the CON cows, paired based on comparable body weight; for the SR group, each cow received 70% self-derived rumen content. The results revealed no significant differences in the thicknesses of the stratum corneum, granulosum, and spinosum/basale layers, as well as the total depth of the epithelium between the SR and DR groups. All these measurements exhibited a decreasing trend and fluctuations over time after the transfer. Notably, these fluctuations tended to stabilize at 13 or 16 d after RCT in the SR group, whereas they tended to stabilize after 8 or 13 d of transfer for the DR group. Transcriptome sequencing revealed that a total of 277 differentially expressed genes (DEGs) were identified between the 2 groups. Enrichment analysis showed that the DEGs were significantly enriched in 11 Gene Ontology biological processes and 14 KEGG pathways. The DEGs corresponding to almost any of these 11 biological process terms and 14 pathways showed mixed up- or downregulation following RCT. Metabolomics analysis indicated that a total of 33 differential metabolites were detected between the SR and DR groups, mainly enriched in 5 key metabolic pathways, including plant polysaccharides and starch degradation, lipid metabolism, amino sugar and nucleotide metabolism, purine metabolism, and Krebs cycle. Among them, the levels of differential metabolites associated with the degradation of plant polysaccharides and starches, metabolism of amino sugars and nucleotides, and purine metabolism pathways were significantly elevated in the DR cows. The results of blood biochemical parameters showed that the triglyceride concentration of the DR cows was increased than that of the SR cows, comparable to the level observed in the CON cows during the SARA induction period. Generally, our findings indicated that RCT facilitated the recovery of rumen epithelial morphological structure but did not promote its function recovery. Moreover, RCT enhanced rumen plant polysaccharide and starch degradation, amino sugar and nucleotide sugar metabolism, as well as purine metabolism. Additionally, it further promoted the recovery of plasma metabolites related to lipid metabolism.
PubMed: 38908691
DOI: 10.3168/jds.2024-24694 -
Archives of Microbiology Jun 2024Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and...
Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus.
Topics: Vibrio parahaemolyticus; Biofilms; Type VI Secretion Systems; Bacterial Proteins; Cyclic GMP; Humans; Gene Expression Regulation, Bacterial; HeLa Cells
PubMed: 38907796
DOI: 10.1007/s00203-024-04060-x -
Nature Communications Jun 2024Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding...
Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.
Topics: Adenosine Triphosphate; Biosensing Techniques; Escherichia coli; Pseudomonas putida; Fatty Acids; Polyhydroxyalkanoates; Energy Metabolism; Carbon; Oleic Acid
PubMed: 38906854
DOI: 10.1038/s41467-024-49579-1 -
Plant Signaling & Behavior Dec 2024Extracellular ATP (eATP) orchestrates vital processes in plants, akin to its role in animals. P2K1 is a crucial receptor mediating eATP effects. Immunoprecipitation...
Extracellular ATP (eATP) orchestrates vital processes in plants, akin to its role in animals. P2K1 is a crucial receptor mediating eATP effects. Immunoprecipitation tandem mass spectrometry data highlighted FERONIA's significant interaction with P2K1, driving us to explore its role in eATP signaling. Here, we investigated putative P2K1-interactor, FERONIA, which is a versatile receptor kinase pivotal in growth and stress responses. We employed a FERONIA loss-of-function mutant, , to dissect its effects on eATP signaling. Interestingly, showed distinct calcium responses compared to wild type, while eATP-responsive genes were constitutively upregulated in . Additionally, displayed insensitivity to eATP-regulated root growth and reduced cell wall accumulation. Together, these results uncover a role for FERONIA in regulating eATP signaling. Overall, our study deepens our understanding of eATP signaling, revealing the intricate interplay between P2K1 and FERONIA impacting the interface between growth and defense.
Topics: Plant Roots; Signal Transduction; Arabidopsis Proteins; Arabidopsis; Adenosine Triphosphate; Gene Expression Regulation, Plant; Phosphotransferases; Protein Serine-Threonine Kinases
PubMed: 38905329
DOI: 10.1080/15592324.2024.2370706