Mycobacterium tuberculosis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA118223 | ENA |
REPOSITORIES: ENA
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Project description:Background The amplification of bacterial RNA is required if complex host-pathogen interactions are to be studied where the recovery of bacterial RNA is limited. Here, using a whole genome Mycobacterium tuberculosis microarray to measure cross-genome representation of amplified mRNA populations, we have investigated two approaches to RNA amplification using different priming strategies. The first using oligo-dT primers after polyadenylation of the bacterial RNA, the second using a set of mycobacterial amplification-directed primers. Results The reproducibility, sensitivity, and the representational bias introduced by these amplification systems were examined by contrasting expression profiles of the amplified products from inputs of 500, 50 and 5ng total M.tuberculosis RNA with unamplified RNA from the same source. In addition a model M.tuberculosis system of microaerophilic growth and non-replicating persistence was used to assess the capability of amplified RNA microarray comparisons from an experimental system where RNA yield is restricted. Mycobacterial RNA was reproducibly amplified using both methods from as little as 5ng total RNA (~ equivalent to 2E+05 bacilli). Differential gene expression patterns observed with unamplified RNA in the switch from aerobic to microaerophilic growth were also reflected in the amplified expression profiles. Conclusions Here we describe two reproducible methods of bacterial RNA amplification that will allow previously intractable host-pathogen interactions during bacterial infection to be explored at the whole genome level by RNA profiling. Data is also available from http://bugs.sgul.ac.uk/E-BUGS-42
2008-03-12 | E-BUGS-42 | biostudies-arrayexpress
Project description:MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate ≤ 1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic shiftdown.
2017-01-25 | GSE89830 | GEO
Project description:Pyrazinamide (PZA) is one of the first line antibiotics used for the treatment of tuberculosis (TB). we have used human monocyte and a mouse model of pulmonary TB to investigate whether treatment with PZA, in addition to its known anti-mycobacterial properties, modulate the host immune response during Mycobacterium tuberculosis (Mtb) infection.
2013-06-28 | GSE48027 | GEO
Project description:The result validated the connections between mutations, gene expression and mycobacterial pathogenicity, and identified new possible virulence-associated pathways in M. tuberculosis.
2022-07-13 | GSE203662 | GEO
Project description:An attenuated strain used in mycobacterial virulence research
| PRJNA18649 | ENA
Project description:We have performred RNA-seq analysis of WT, ∆phoP and ∆sigH Mtb H37Rv under normal and redox (Diamide) stress, to investigate the role of PhoP and SigH in maintaning the redox homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. Briefly, 25 ml of bacterial culture was grown to mid-log phase (OD600= 0.4 to 0.6) and combined with 40 ml of 5 M guanidinium thiocyanate solution containing 1% β-mercaptoethanol and 0.5% Tween 80. Cells were pelleted by centrifugation, and lysed by re-suspending in 1 ml Trizol (Ambion) in the presence of Lysing Matrix B (100 µm silica beads; MP Bio) using a FastPrep-24 bead beater (MP Bio) at a speed setting of 6.0 for 30 seconds. The procedure was repeated for 2-3 cycles with incubation on ice in between pulses. Next, cell lysates were centrifuged at 13000 rpm for 10 minutes; supernatant was collected and processed for RNA isolation using Direct-ZolTM RNA isolation kit (ZYMO) as per manufacturer’s recommendation. Following extraction, RNA was treated with DNAse I (Promega) to degrade contaminating DNA, and integrity was assessed using a Nanodrop (ND-1000, Spectrophotometer). RNA samples were further checked for intactness of 23S and 16S rRNA using formaldehyde-agarose gel electrophoresis, and Qubit fluoremeter (Invitrogen). RNA integrity was checked using Agilent 2200 Tape Station system (Agilent Technologies). Library construction, RNA-sequencing and data analysis have been carried out by Agrigenome Labs Private Limited (Cochin), India. The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Although recent evidence suggests that at low pH M. tuberculosis encounters reductive stress, the mechanism of integrated regulation of stress response remains unknown. Unexpectedly, we find that PhoP contributes to mycothiol level and a PhoP-depleted bacilli shows enhanced susceptibility to redox stress. Because SigH is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. Our results suggest that while PhoP controls pH homeostasis via its interaction with SigE, SigH-dependent PhoP expression, but not PhoP -SigH interaction, and direct recruitment of SigH, but not PhoP controls expression of mycobacterial thioredoxin genes. Together, these results uncover novel stress-specific interaction events of sigma factors and PhoP or lack thereof, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol homeostasis. The main purpose of this study is to understand how mycobacteria can sense numerous stress conditions and mount an appropriate stress response. Although recent evidence suggests that at low pH M. tuberculosis encounters reductive stress, the mechanism of integrated regulation of stress response remains unknown. Unexpectedly, we find that PhoP contributes to mycothiol level and a PhoP-depleted bacilli shows enhanced susceptibility to redox stress. Because SigH is known to control expression of redox inducible genes, we probed whether previously-reported PhoP-SigH interaction accounts for mycobacterial redox stress response. Our results suggest that while PhoP controls pH homeostasis via its interaction with SigE, SigH-dependent PhoP expression, but not PhoP -SigH interaction, and direct recruitment of SigH, but not PhoP controls expression of mycobacterial thioredoxin genes. Together, these results uncover novel stress-specific interaction events of sigma factors and PhoP or lack thereof, as the underlying mechanisms of an adaptive programme, which couples low pH conditions and mycobacterial thiol homeostasis.
2022-04-09 | GSE171775 | GEO
Project description:A novel phenotypic drug screen identifies potent inhibitors of mycobacterial virulence protein secretion
| PRJNA251986 | ENA
Project description:Virulence effectors secreted by Mycobacterium tuberculosis (Mtb) help subvert host immune mechanisms and, therefore, are critical for establishing infection and pathogenesis. However, knowledge in terms of signaling mechanisms that modulate the secretion of virulence factors is sparse. We performed high-throughput secretome, phosphoproteome, and phospho-secretome analysis of Mtb. We combined the analysis with empirical validations to show regulation of mycobacterial secretion through protein phosphorylation. System level PPI network analysis superimposed with the secretome, phosphoproteome, and phospho-secretome profile revealed an intricate relationship between phosphorylation and secretion. At the core of the network was a key virulence factor CFP10. We identified PknA to be the kinase responsible for phosphorylating CFP10. Using genetic tools, we show that phosphomimetic mutation of CFP10 negatively regulates the secretion of virulence mediator ESAT6. Significantly, the dynamics of CFP10 phosphorylation strongly influenced bacterial virulence and survival within macrophages and mice. Together, the results show that the dynamic phosphorylation status of the secretory protein CFP10 regulates the secretion of virulence factors and impacts virulence.
2023-09-22 | PXD045586 | Pride
Project description:The immunomodulatory mycobacterial surface antigen lipoarabinomannan (LAM) with its immunogenic glycan component arabinomannan (AM) facilitates Mycobacterium tuberculosis’ (Mtb) escape from the host’s immune response. Some but not all antibodies against AM can protect against TB. To better understand which of AM’s structures to target, we must first identify the spectrum of its epitopes. Through isolating and characterizing a panel of novel human mAbs with binding to distinct AM OS motifs, we further defined the glycan epitope structures, identified a new epitope, and determined their differences among mycobacterial strains.
2024-06-01 | GSE263497 | GEO
Project description:Mycobacterium tuberculosis (Mtb) secretes pathogenicity factors and immunologically active molecules via membrane vesicles. However, nothing is known about the mechanisms involved in mycobacterial vesicle biogenesis. This study investigates molecular determinants of membrane vesicle production in Mtb by analyzing Mtb cells under conditions of high vesicle production: iron limitation and VirR restriction. Ultrastructural analysis showed extensive cell envelope restructuring in association with vesicle release that correlated with downregulation of cell surface lipid biosynthesis and peptidoglycan alterations. Comparative transcriptomics showed common upregulation of the iniBAC operon in association with high vesicle production in Mtb cells. Vesicle production analysis demonstrated that the dynamin-like proteins (DLPs) encoded by this operon, IniA and IniC, are necessary for release of EV by Mtb in culture and in infected macrophages. Isoniazid, a first-line antibiotic, used in tuberculosis treatment, was found to stimulate vesicle release in a DLP-dependent manner. Our results provide a new understanding of the function of mycobacterial DLPs and mechanistic insights into vesicle biogenesis. The findings will enable further understanding of the relevance of Mtb-derived extracellular vesicles in the pathogenesis of tuberculosis and may open new avenues for therapeutic research.
2023-06-22 | GSE143996 | GEO