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Inducible knockdown of Plasmodium gene expression using the glmS ribozyme

ABSTRACT: Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3M-bM-^@M-2 untranslated region (3M-bM-^@M-2-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery. mRNA profiles were generated from 3D7 wild-type and DHFR-TS-GFP_glmS integrant parasites in untreated and treated with 10 mM Glucosamine conditions in duplicate.

ORGANISM(S): Plasmodium falciparum

SUBMITTER: Philip Shaw

PROVIDER: E-GEOD-43125 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Inducible knockdown of Plasmodium gene expression using the glmS ribozyme

Project description:Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region (3′-UTR) are efficiently knocked down in transgenic P. falciparum parasites in response to exogenous glucosamine (GlcN) inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following GlcN treatment. GlcN-induced ribozyme activation also led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). GlcN treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme is thus an important tool for study of P. falciparum essential genes and anti-malarial drug discovery.

2013-09-03 | GSE43125 | GEO

Plasmodium falciparum MORC protein modulates gene expression through interaction with heterochromatin

Project description:The MORC (microrchidia) family of proteins is highly conserved in all eukaryotic cells and have been shown to play diverse roles by forming protein-protein interactions with immune-responsive proteins, SWI chromatin remodeling complexes, histone deacetylases, and histone tail modifications across metazoans. To dissect the functional roles of MORC in the human malaria parasite, Plasmodium falciparum, we developed a glmS based ribozyme knockdown system to induce PfMORC knockdown upon glucosamine (GlcN) induction. We further conducted a transcriptomic analysis in PfMORC-HA-glmS knockdown parasites at the asexual stage to investigate alterations in global gene expression. Our results indicate an overrepresentation of downregulated genes belonging to the heterochromatin-associated hypervariable gene family proteins. Overall, we found that PfMORC controls the asexual gene expression of the P. falciparum and can be a potential target for malaria disease containment.

2023-11-15 | GSE241313 | GEO

RNA-seq of C.elegans and M.musculus in the presence and absence of D-Glucosamine (GlcN)

Project description:D-Glucosamine (2-amino-2-deoxy-D-glucose, C.A.S.# 3416-24-8) (GlcN) is a freely available and commonly used dietary supplement possibly promoting cartilage health in humans which also acts as an inhibitor of glycolysis. We here find that GlcN extends C. elegans lifespan by impairing glucose metabolism to activate AMP-activated protein kinase (AMPK/AAK2) leading to increased mitochondrial biogenesis. Consistent with the concept of mitohormesis, this promotes increased formation of mitochondrial reactive oxygen species (ROS) and p38/PMK-1-mediated stress signaling culminating in increased expression of the nematodal amino acid-transporter 1 (aat-1) gene. Ameliorating mitochondrial ROS formation as well as impairment of aat-1-expression abolishes GlcN-mediated lifespan extension in a NRF2/SKN-1-dependent fashion. Notably and unlike other calorie restriction mimetics (CRM) like 2-deoxy-D-glucose (2DG, DOG), GlcN extends lifespan of aging C57BL/6 mice (log-rank: p=0.002; cox regression: p=0.01) similarly paralleled by an induction of mitochondrial biogenesis, increased expression of several murine amino acid transporters, as well as increased amino-acid catabolism. Taken together, GlcN mimics a ketogenic diet to extend healthspan in evolutionary distinct species. 24 samples: 12 mRNA profiles of C.elegans: 6 without GlcN and 6 with GlcN supplementaion; 12 mRNA profiles of M.musculus: 6 without GlcN and 6 with GlcN supplementaion

2014-03-07 | E-GEOD-54853 | biostudies-arrayexpress

Expression data from glucosamine-treated human synovial MH7A cells

Project description:Osteoarthritis (OA) is one of the major joint diseases. Glucosamine (GlcN) is widely used as a dietary supplement for OA. However, its precise mechanisms are not well understood. GlcN is utilized for the O-linked-N-acetylglucosamine (O-GlcNAc) modification of proteins, which participates in the regulation of cellular functions. In this study, we comprehensively analyzed the effect of GlcN and alloxan (O-GlcNAc transferase inhibitor, which prevented O-GlcNAc modification) on the gene expression using DNA microarray. GlcN downregulated or upregulated genes. Furthermore, among the GlcN-downregulated or upregulated genes, the expression of 62.7% of the genes was restored by alloxan.. Thus, it is suggested that GlcN regulates the gene expression by an O-GlcNAc modification-dependent or -independent mechanism.

2016-11-07 | GSE72575 | GEO

The ROK-family regulator RokL6 is involved in GlcN toxicity by controlling the transcription of SCO1448 in Streptomyces coelicolor [ChIP-seq]

Project description:Streptomycetes are saprophytic bacteria that grow on complex polysaccharides, such as cellulose, starch, chitin and chitosan. For the monomeric building blocks glucose, maltose and N-acetylglucosamine the metabolic pathway is well documented, but that of glucosamine (GlcN) is currently unknown. Importantly, GlcN is lethal to Streptomyces coelicolor nagB mutants, which lack glucosamine-6-phosphate deaminase activity. Here we report that spontaneous and directed mutations in the gene for the ROK-family protein RokL6 (SCO1447) relieve GlcN toxicity in nagB mutants of S. coelicolor. RNA sequencing, ChIP-Seq and over-expression studies revealed that RokL6 acts by directly repressing SCO1448, which encodes a sugar exporter; RokL6 thereby only binds to the rokL6-SCO1448 intergenic region in vivo, with consensus binding site C(T)TATCAGG - 7 nt - CCTGATAG(A). The exact transcriptional start sites for rokL6 and SCO1448 were determined using 5’RACE. RokL6 represses the transcription of both rokL6 and SCO1448 by binding to overlapping promoter sequences. Taken together, our data show that RokL6 and SCO1448 are novel GlcN-related genes, whereby RokL6 directly controls the transcription of SCO1448. The latter is a key protein in the defense of S. coelicolor against the toxicity of GlcN in a nagB-mutant background, most likely via the export of GlcN-derived toxic intermediates.

2023-06-19 | GSE234438 | GEO

The ROK-family regulator RokL6 is involved in GlcN toxicity by controlling the transcription of SCO1448 in Streptomyces coelicolor [RNA-seq]

2023-06-19 | GSE234437 | GEO

RNA-seq of C.elegans and M.musculus in the presence and absence of D-Glucosamine (GlcN)

2014-03-07 | GSE54853 | GEO

Glucosamine facilitates cardiac ischemic recovery via recruiting Ly6Clow monocytes in a STAT1 O-GlcNAcylation dependent fashion

Project description:The O-linked β-N-acetylglucosamine (O-GlcNAc) modification on intracellular proteins controls diverse biological processes. Though sustained hyper-O-GlcNAcylation aggravates pathogenesis of many chronic diseases, accumulating evidence also indicates that acute augmentation in O-GlcNAcylation seems to benefit disease healing in some cases. Glucosamine (GlcN) is a freely available and commonly used dietary supplement for human cartilage health, which also activates the hexosamine biosynthesis pathway and induces protein O-GlcNAcylation. Here we show that both GlcN early and late therapies effectively facilitate cardiac recovery in mice by elevating accumulation of Ly6Clow Mo/Mps in infarcted hearts. Eliminating Mo/Mps with clodronate liposomes fully abolishes aforementioned cardiac healing role of GlcN. Importantly, GlcN supplementation accelerates not only in vitro mobility of reparative Mo/Mps but also in vivo infiltration of Ly6Clow Mos. Mechanistically, GlcN positively regulates transcription of myeloid CX3C chemokine receptor 1 (Cx3cr1) by improving STAT1 O-GlcNAcylation, which stabilizes STAT1 and subsequently promotes chemotaxis and infiltration of Ly6Clow Mos. In summary, we identify a novel anti-inflammatory role of GlcN and suggest that its protective effects are mediated through chemotaxis of Ly6Clow Mos in a STAT1/CX3CR1-dependent fashion, which is controlled by O-GlcNAcylation of STAT1. Our study provides a novel clue for Mo/Mps modulator therapies aimed at reducing post-MI hyperinflammation in ischemic myocardium.

2022-04-06 | GSE193613 | GEO

AMINO SUGARS RESHAPE INTERACTIONS BETWEEN STREPTOCOCCUS MUTANS AND STREPTOCOCCUS GORDONII

Project description:Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are abundant carbon and nitrogen sources that are continually supplied in host secretions and the diet to biofilms colonizing the human mouth. Evidence is emerging that these amino sugars may provide an ecological advantage to beneficial commensals over oral 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 bacterium when they were cultured alone. Likewise, co-cultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different than the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism, in both 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 supernates. Differing a previous report, growth of S. mutans alone with GlcN inhibited expression of multiple operons required for mutacin production. Co-cultivation with S. gordonii consistently increased the expression by S. mutans of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes. 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 co-cultures. In conclusion, amino sugars profoundly altered the interactions between the pathogen and the commensal, likely by reprogramming their central metabolism.

2020-06-09 | GSE152021 | GEO

Molecular characterization of Plasmodium falciparum Bruno/CELF RNA binding proteins

Project description:The human malaria parasite Plasmodium falciparum employs intricate post-transcriptional regulatory mechanisms in different stages of its life cycle. Despite the importance of post-transcriptional regulation, key elements of these processes, namely RNA binding proteins (RBPs), are poorly characterized. In this study, the RNA binding properties of P. falciparum proteins were characterized including two putative members of the Bruno/CELF family of RBPs (PfCELF1 and PfCELF2), dihydrofolate reductase-thymidylate synthase (PfDHFR-TS), and adenosine deaminase (PfAda).The mRNA targets of these P. falciparum proteins were investigated by ribonomics using DNA microarrays.

2014-10-23 | GSE56329 | GEO

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