Involvement of transcription elongation factor GreA in Mycobacterium tuberculosis viability, antibiotic susceptibility, and intracellular fitness
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ABSTRACT:
PROVIDER: PRJNA601528 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10901459_1.fastq.gz | Fastqsanger.gz | |||
| SRR10901459_2.fastq.gz | Fastqsanger.gz | |||
| SRR10901460_1.fastq.gz | Fastqsanger.gz | |||
| SRR10901460_2.fastq.gz | Fastqsanger.gz | |||
| SRR10901461_1.fastq.gz | Fastqsanger.gz |
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Project description:There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (?greA) was generated in Mycobacterium smegmatis, Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardation and poor survival in response to adverse stress, besides rendering M. tuberculosis more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting greA results in the differential regulation of 195 genes in M. smegmatis with 167 being negatively regulated. Among these, KEGG pathways significantly enriched for differentially regulated genes included tryptophan metabolism, starch and sucrose metabolism, and carotenoid biosynthesis, supporting a role of GreA in the metabolic regulation of mycobacteria. Moreover, like Escherichia coli GreA, M. smegmatis GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of glgX, a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of glgX by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of greA. Our data indicates that GreA is an important factor for the survival and resistance establishment in Mycobacterium spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment.
| S-EPMC7104715 | biostudies-literature
Project description:To understand how GreA may affect gene expression at the transcriptional level, we performed whole-transcriptome RNA-seq analyses. To examine whether GreA binds to specific chromosomal regions, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and then sought to verify the binding mode of GreA. ChIP-seq experiments were performed by using M. smegmatis strains producing GreA proteins fused with two repeats of the FLAG epitope (FLAG2). GreA-FLAG2-DNA nucleoprotein complexes fixed with formaldehyde in the log phase of growth were immunoprecipitated using magnetic beads. To exclude unspecific interactions with magnetic beads, we used a WT strain lacking the FLAG epitope as a negative control for our ChIP-seq experiments. Enriched regions were determined by comparison to the background noise level, which was estimated versus the input DNA of each ChIP-Seq replicate. Disrupting greA resulted in the differential regulation of 195 genes in M. smegmatis during the exponential growth. Among these, 28 were positively regulated and 167 were negatively regulated. The identified GreA-FLAG2 binding sites included 1005 ChIP-seq peaks that were confirmed in two biological replicates and absent in WT strain.
2020-01-16 | GSE143764 | GEO
Project description:The involvement of Mycobacterium type III-A CRISPR-Cas system in oxidative stress and intracellular fitness
| PRJNA699439 | ENA
Project description:Intracellular Ca2+ release through the Inositol 1,4,5-Triphosphate Receptor (InsP3R) is a feature of all multicellular organisms in which it shapes the temporal and spatial aspects of calcium signaling in cells, affecting several developmental and physiological processes. Mutants in the InsP3R gene of Drosophila (itpr) exhibit a range of defects which include growth defects and larval lethality.The Drosophila Insulin-like peptides (ILPs) are encoded by multigene families that are expressed in the brain and other tissues. Upon secretion, these peptides likely serve as hormones, neurotransmitters, and growth factors. In Drosophila melanogaster, molecular genetic studies have revealed Drosophila ILPs as elements of a conserved insulin signaling pathway, and as in other animal models, it appears to play a key role in metabolism, growth, reproduction, and aging. Previous work from our group has shown that InsP3R function can be rescued by ectopically expressing InsP3R in dilp cells or can be compensated by altering the activity of Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA). To understand the molecular basis of larval lethality and growth defect in Drosophila InsP3R mutants and to decipher possible cross talk of itpr gene with other signaling pathways we have carried out genome wide microarray experiments. We have compared the transcript profiles of i) itpr mutant larvae and wild type larvae, ii) dilp rescue larvae with itpr mutant larvae iii) itpr and SERCA double mutant larvae with itpr mutant larvae. By this we have identified genes whose levels are altered in itpr mutants and are restored towards wild type in dilp rescue larvae with itpr mutant larvae and itpr and SERCA double mutants. Our experiments help in identify genes that are regulated by changes in intracellular Ca2+ in the context of larval viability may contribute to the better understanding of the major human pathologies caused by calcium misbalance or by dysregulation of the insulin/IGF pathway. The 8x60k array AMADID: 27326 slide was scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Three were used with Mutant, three for Dilp Rescue and two for Kum Rescue experiments . The data was analysed by GeneSpring GX Agilent . The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from biological triplicates for Mutant, biological treplicates for Dilp Rescue and biological duplicates for Kum Rescue.
2012-06-01 | E-GEOD-29736 | biostudies-arrayexpress
Project description:Previous studies suggest that type I and type II CRISPR-Cas systems were employed to evade host immunity by targeting interference of bacteria’s own genes. Although Mycobacterium bovis (M. bovis) and Mycobacterium tuberculosis (M. tuberculosis) possess integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes (csm2~6, cas10, cas6) were significantly upregulated under oxidative stress treatment. To explore the function of type III-A CRSIRP-Cas system, Total CRISPR system (TCR) mutant strain was generated in M. bovis Bacille Calmette-Guérin (BCG). Deletion of TCR results in increased sensitivity in response to H2O2 and reduced cell envelope integrity. By using RNA-Seq, 590 differential expression genes were found in mutant for TCR, of which 220 genes were upregulated and 370 genes were downregulated, indicating an important role of TCR in controlling gene expression in mycobacteria. Consistently, disrupting TCR results in poor intracellular survival in vivo and in vitro. Moreover, we showed for the first time that TCR contributed to the regulation of regulatory T cell population, supporting a role of TCR in modulating host immunity. These observations demonstrate that type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, may be a potential target for therapy.
2021-02-05 | GSE166137 | GEO
Project description:Intracellular Ca2+ release through the Inositol 1,4,5-Triphosphate Receptor (InsP3R) is a feature of all multicellular organisms in which it shapes the temporal and spatial aspects of calcium signaling in cells, affecting several developmental and physiological processes. Mutants in the InsP3R gene of Drosophila (itpr) exhibit a range of defects which include growth defects and larval lethality.The Drosophila Insulin-like peptides (ILPs) are encoded by multigene families that are expressed in the brain and other tissues. Upon secretion, these peptides likely serve as hormones, neurotransmitters, and growth factors. In Drosophila melanogaster, molecular genetic studies have revealed Drosophila ILPs as elements of a conserved insulin signaling pathway, and as in other animal models, it appears to play a key role in metabolism, growth, reproduction, and aging. Previous work from our group has shown that InsP3R function can be rescued by ectopically expressing InsP3R in dilp cells or can be compensated by altering the activity of Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA). To understand the molecular basis of larval lethality and growth defect in Drosophila InsP3R mutants and to decipher possible cross talk of itpr gene with other signaling pathways we have carried out genome wide microarray experiments. We have compared the transcript profiles of i) itpr mutant larvae and wild type larvae, ii) dilp rescue larvae with itpr mutant larvae iii) itpr and SERCA double mutant larvae with itpr mutant larvae. By this we have identified genes whose levels are altered in itpr mutants and are restored towards wild type in dilp rescue larvae with itpr mutant larvae and itpr and SERCA double mutants. Our experiments help in identify genes that are regulated by changes in intracellular Ca2+ in the context of larval viability may contribute to the better understanding of the major human pathologies caused by calcium misbalance or by dysregulation of the insulin/IGF pathway.
2012-06-02 | GSE29736 | GEO
Project description:Intracellular Staphylococcus aureus persisters upon antibiotic exposure
| PRJNA586837 | ENA
Project description:Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here we provide in vitro evidence of Staphylococcus aureus persisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. Intracellular S. aureus persisters remain metabolically active, but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell-wall stress, SOS and heat-shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellular S. aureus persisters may constitute a reservoir for relapsing infection, and could contribute to therapeutic failures.
2020-03-25 | GSE139659 | GEO
Project description:Some intracellular bacteria are known to cause long-term infections for periods of time that last decades without compromising the viability of the host. Although of critical importance, the changes that intracellular bacteria suffer during this long process of residence in a host cell environment remain obscure. Here, we report an experimental approach to study the adaptations of intracellular mycobacteria forced by a long-term intracellular lifestyle. Long-term infection of host macrophages with mycobacteria was maintained for a period of years. Mycobacteria in the long-term infected macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, preference for glucose as a carbon source and neutral lipids accumulation. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states of bacteria living in fluctuating intracellular environments.
2014-12-31 | GSE49976 | GEO
Project description:BRCA1 mutational complementation induces synthetic viability
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