GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.
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ABSTRACT:
SUBMITTER: Dos Reis TF
PROVIDER: S-EPMC6812930 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Experiment design Type of experiment: Gene expression profiling analysis of S.cerevisiae W303-1a. Experimental factor: genetic variation (WT, gpr1 and gpa2 mutant strains). Number of hybridizations: 6. Single-colour labeling was performed for each sample. Sample preparation: Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturers' instructions. Labeling protocol 10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Hybridization protocol 10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was controled by use of the GeneChip™ Eukaryotic Hybridization Control Kit (Affymetrix). Washing and staining was performed in a Fluidics Station 400 (Affymetrix) using the protocol EukGE-WS2v4. Measurement data, specifications and data processing Scanning was performed in an Affymetrix GeneChip scanner. Chip analysis was performed using the Affymetrix Microarray Suite v5. Changes in gene expression were assessed by looking for concordant changes between all replicates using a signed Wilcoxon rank test. The “change” p-value threshold was < 0.025 for increase and > 0.975 for decrease. Array design Microarray analysis was performed using YGS98 GeneChips™ oligonucleotide arrays (Affymetrix #900256) representing approximately 6000 ORFs. Sequences and GenBank accession numbers of all probe sets are available at the Affymetrix home page http://www.affymetrix.com/index.affx. Keywords: ordered
2004-12-08 | GSE970 | GEO
Project description:No information is available in the literature about the influence of temperature (T) on Penicillium and Aspergillus spp. growth and mycotoxin production on cheese rinds. The aim of this work was to: (i) study fungal ecology on cheese in terms of T requirements, focusing on the partitioning of mycotoxins between the rind and mycelium; and (ii) validate predictive models previously developed by in vitro trials. Grana cheese rind blocks were inoculated with A. versicolor, P. crustosum, P. nordicum, P. roqueforti, and P. verrucosum, incubated at different T regimes (10-30 °C, step 5 °C) and after 14 days the production of mycotoxins (ochratoxin A (OTA); sterigmatocystin (STC); roquefortine C (ROQ-C), mycophenolic acid (MPA), Pr toxin (PR-Tox), citrinin (CIT), cyclopiazonic acid (CPA)) was quantified. All the fungi grew optimally around 15-25 °C and produced the expected mycotoxins (except MPA, Pr-Tox, and CIT). The majority of the mycotoxins produced remained in the mycelium (~90%) in three out of five fungal species (P. crustosum, P. nordicum, and P. roqueforti); the opposite occurred for A. versicolor and P. verrucosum with 71% and 58% of STC and OTA detected in cheese rind, respectively. Available predictive models fitted fungal growth on the cheese rind well, but validation was not possible for mycotoxins because they were produced in a very narrow T range.
| S-EPMC7022280 | biostudies-literature
Project description:Modulation of energy metabolism to a highly glycolytic phenotype, i.e. Warburg effect, is a common phenotype of cancer and activated immune cells allowing increased biomass-production for proliferation and cell division. Endoplasmic reticulum (ER)-localized ADP-dependent glucokinase (ADPGK) has been shown to play a critical role in T cell receptor activation-induced remodeling of energy metabolism, however the underlying mechanisms remain unclear. Therefore, we established and characterized in vitro and in vivo models for ADPGK-deficiency using Jurkat T cells and zebrafish. Upon activation, ADPGK knockout Jurkat T cells displayed increased cell death and ER stress. The increase in cell death resulted from a metabolic catastrophe and knockout cells displayed severely disturbed energy metabolism hindering induction of Warburg phenotype. ADPGK knockdown in zebrafish embryos led to short, dorsalized body axis induced by elevated apoptosis. ADPGK hypomorphic zebrafish further displayed dysfunctional glucose metabolism. In both model systems loss of ADPGK function led to defective N- and O-glycosylation. Overall, our data illustrate that ADPGK is part of a glucose sensing system in the ER modulating metabolism via regulation of N- and O-glycosylation.
| S-EPMC6776650 | biostudies-literature
Project description:How glucose sensing by the nervous system impacts the regulation of ? cell mass and function during postnatal development and throughout adulthood is incompletely understood. Here, we studied mice with inactivation of glucose transporter 2 (Glut2) in the nervous system (NG2KO mice). These mice displayed normal energy homeostasis but developed late-onset glucose intolerance due to reduced insulin secretion, which was precipitated by high-fat diet feeding. The ? cell mass of adult NG2KO mice was reduced compared with that of WT mice due to lower ? cell proliferation rates in NG2KO mice during the early postnatal period. The difference in proliferation between NG2KO and control islets was abolished by ganglionic blockade or by weaning the mice on a carbohydrate-free diet. In adult NG2KO mice, first-phase insulin secretion was lost, and these glucose-intolerant mice developed impaired glucagon secretion when fed a high-fat diet. Electrophysiological recordings showed reduced parasympathetic nerve activity in the basal state and no stimulation by glucose. Furthermore, sympathetic activity was also insensitive to glucose. Collectively, our data show that GLUT2-dependent control of parasympathetic activity defines a nervous system/endocrine pancreas axis that is critical for ? cell mass establishment in the postnatal period and for long-term maintenance of ? cell function.
| S-EPMC3871223 | biostudies-literature
Project description:Despite vaccines, Streptococcus pneumoniae kills more than a million people yearly. Thus, understanding how pneumococci transition from commensals to pathogens is particularly relevant. Quorum sensing regulates collective behaviors and thus represents a potential driver of commensal-to-pathogen transitions. Rgg/small hydrophobic peptide (SHP) quorum-sensing systems are widespread in streptococci, yet they remain largely uncharacterized in S. pneumoniae. Using directional transcriptome sequencing, we show that the S. pneumoniae D39 Rgg0939/SHP system induces the transcription of a single gene cluster including shp and capsule gene homologs. Capsule size measurements determined by fluorescein isothiocyanate-dextran exclusion allowed assignment of the system to the regulation of surface polysaccharide expression. We found that the SHP pheromone induced exopolysaccharide expression in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the Rgg system resulted in a mutant with increased capsule size. In line with previous studies showing that capsule expression is inversely associated with biofilm formation, we found that biofilm formed on lung epithelial cells was decreased in the overexpression strain and increased in an rgg deletion mutant. Although no significant differences were observed between D39 and the rgg deletion mutant in a mouse model of lung infection, in competitive assays, overexpression reduced fitness. This is the first study to reveal a quorum-sensing system in streptococci that regulates exopolysaccharide synthesis from a site distinct from the original capsule locus. IMPORTANCE Quorum sensing regulates bacterial social behaviors by production, secretion, and sensing of pheromones. In this study, we characterized a new quorum-sensing system of the Rgg/SHP class in S. pneumoniae D39. The system was found to directly induce the expression of a single gene cluster comprising the gene for the SHP pheromone and genes with putative functions in capsule synthesis. Capsule size, as measured by dextran exclusion, was increased by SHP exposure in R36A, an unencapsulated derivative of D39. In the encapsulated parent strain, overexpression of the gene cluster increased capsule size, supporting the role of Rgg/SHP in the synthesis of surface polysaccharides. Further, we found that biofilm formation on epithelial cells was reduced by overexpression of the system and increased in a mutant with an rgg deletion. Placing surface polysaccharide expression under quorum-sensing regulation may enable S. pneumoniae to tune interactions with the host and other bacteria in accordance with environmental and cell density conditions.
| S-EPMC5597970 | biostudies-literature
Project description:G-protein-coupled receptors (GPCRs) are the largest and most diverse group of membrane receptors in eukaryotes and detect a wide array of cues in the human body. Here we describe a molecular device that couples CRISPR-dCas9 genome regulation to diverse natural and synthetic extracellular signals via GPCRs. We generate alternative architectures for fusing CRISPR to GPCRs utilizing the previously reported design, Tango, and our design, ChaCha. Mathematical modeling suggests that for the CRISPR ChaCha design, multiple dCas9 molecules can be released across the lifetime of a GPCR. The CRISPR ChaCha is dose-dependent, reversible, and can activate multiple endogenous genes simultaneously in response to extracellular ligands. We adopt the design to diverse GPCRs that sense a broad spectrum of ligands, including synthetic compounds, chemokines, mitogens, fatty acids, and hormones. This toolkit of CRISPR-coupled GPCRs provides a modular platform for rewiring diverse ligand sensing to targeted genome regulation for engineering cellular functions.
| S-EPMC5738360 | biostudies-literature
Project description:Intestinal glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion and acts as a neuropeptide to control glucose homeostasis, but little is known whether intestinal GLP-1 has any effect in the control of hepatic glucose production (HGP). Here we found that intraduodenal infusion of GLP-1 activated duodenal PKC-?, lowered HGP and was accompanied by a decrease in hepatic expression of gluconeogenic enzymes and an increase in hepatic insulin signaling in rats. However, gut co-infusion of either the GLP-1 receptor antagonist Ex-9, or the PKC-? inhibitor rottlerin with GLP-1, negated the ability of gut GLP-1 to lower HGP and to increase hepatic insulin signaling during clamps. The metabolic and molecular signal effects of duodenal GLP-1 were also negated by co-infusion with tetracaine, pharmacologic inhibition of N-methyl-d-aspartate receptors within the dorsalvagal complex, or hepatic vagotomy in rats. In summary, we identified a neural glucoregulatory function of gut GLP-1 signaling.
| S-EPMC5386475 | biostudies-literature
Project description:Purple prairie clover (PPC; Dalea purpurea Vent.) containing 84.5 g/kg dry matter (DM) of condensed tannin (CT) was ensiled without (control) or with polyethylene glycol (PEG) for 76 days, followed by 14 days of aerobic exposure. Changes in fermentation characteristics were determined, and the composition of bacterial and fungal communities were assessed using metagenomic sequencing. The addition of polyethylene glycol (PEG) that deactivated CT at ensiling increased (P < 0.05 to ∼0.001) soluble N, nonprotein N, lactic acid, total volatile fatty acids, ammonia N, deoxynivalenol (DON), and ochratoxin A (OTA) but decreased (P < 0.001) pH and water-soluble carbohydrates. The concentrations of DON and OTA increased (P < 0.001) for both silages, with the extent of increase being greater for control than for PEG-treated silage during aerobic exposure. The PEG-treated silage exhibited higher (P < 0.01 to ∼0.001) copy numbers of total bacteria, Lactobacillus, yeasts, and fungi than the control. The addition of PEG decreased (P < 0.01) bacterial diversity during both ensiling and aerobic exposure, whereas it increased (P < 0.05) fungal diversity during aerobic exposure. The addition of PEG at ensiling increased (P < 0.05) the abundances of Lactobacillus and Pediococcus species but decreased (P < 0.01) the abundances of Lactococcus and Leuconostoc species. Filamentous fungi were found in the microbiome at ensiling and after aerobic exposure, whereas Bacillus spp. were the dominate bacteria after aerobic exposure. In conclusion, CT decreased protein degradation and improved the aerobic stability of silage. These desirable outcomes likely reflect the ability of PPC CT to inhibit those microorganisms involved in lowering silage quality and in the production of mycotoxins.IMPORTANCE The present study reports the effects of condensed tannins on the complex microbial communities involved in ensiling and aerobic exposure of purple prairie clover. This study documents the ability of condensed tannins to lower mycotoxin production and the associated microbiome. Taxonomic bacterial community profiles were dominated by Lactobacillales after fermentation, with a notable increase in Bacillus spp. as a result of aerobic exposure. It is interesting to observe that condensed tannins decreased bacterial diversity during both ensiling and aerobic exposure but increased fungal diversity during aerobic exposure only. The present study indicates that the effects of condensed tannins on microbial communities lead to reduced lactic acid and total volatile fatty acid production, proteolysis, and mycotoxin concentration in the terminal silage and improved aerobic stability. Condensed tannins could be used as an additive to control unfavorable microbial development and maybe enhanced feed safety.
| S-EPMC5812926 | biostudies-literature
Project description:Streptococcus pneumoniae is the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. The luxS-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control of S. pneumoniae biofilm formation. Our results demonstrate that the luxS gene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. The luxS gene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulating S. pneumoniae biofilms, at 24 h postinoculation, two different D39ΔluxS mutants produced ∼80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains with luxS, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39ΔluxS. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels of lytA, which encodes an autolysin previously implicated in biofilms, and also the transcript levels of ply, which encodes the pneumococcal pneumolysin. In conclusion, the luxS-controlled QS system is a key regulator of early biofilm formation by S. pneumoniae strain D39.
| S-EPMC3187250 | biostudies-literature
Project description:The model fungus Aspergillus nidulans synthesizes numerous secondary metabolites, including sterigmatocystin (ST). The production of this toxin is positively controlled by the global regulator veA. In the absence of veA (ΔveA), ST biosynthesis is blocked. Previously, we performed random mutagenesis in a ΔveA strain and identified revertant mutants able to synthesize ST, among them RM1. Complementation of RM1 with a genomic library revealed that the mutation occurred in a gene designated as cpsA. While in the ΔveA genetic background cpsA deletion restores ST production, in a veA wild-type background absence of cpsA reduces and delays ST biosynthesis decreasing the expression of ST genes. Furthermore, cpsA is also necessary for the production of other secondary metabolites, including penicillin, affecting the expression of PN genes. In addition, cpsA is necessary for normal asexual and sexual development. Chemical and microscopy analyses revealed that CpsA is found in cytoplasmic vesicles and it is required for normal cell wall composition and integrity, affecting adhesion capacity and oxidative stress sensitivity. The conservation of cpsA in Ascomycetes suggests that cpsA homologs might have similar roles in other fungal species.
| S-EPMC5506848 | biostudies-literature