Structure of an endogenous mycobacterial MCE lipid transporter
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Mycobacterium Smegmatis Str. Mc2 155 (ncbitaxon:246196)
SUBMITTER: 
Beatrix Ueberheide
PROVIDER: MSV000090807 | MassIVE | Tue Nov 29 13:12:00 GMT 2022
SECONDARY ACCESSION(S): PXD038456
REPOSITORIES: MassIVE
Similar Datasets
Project description:Mycobacterium tuberculosis has the ability to survive inside macrophages under acid-nitrosative stress. M. tuberculosis Rv1685c and its orthologue in M. smegmatis, MSMEG_3765, are induced on exposure to acid-nitrosative stress. Both genes are annotated as TetR transcriptional regulators, a family of proteins that regulate a wide range of cellular activities, including multidrug resistance, carbon catabolism and virulence. Here we demonstrate that MSMEG_3765 is co-transcribed with the upstream genes MSMEG_3762 and MSMEG_3763, encoding efflux pump components. RTq-PCR and GFP-reporter assays showed that the MSMEG_3762/63/65 gene cluster, and the orthologous region in M. tuberculosis (Rv1687c/86c/85c), was up-regulated in a MSMEG_3765 null mutant, suggesting that MSMEG_3765 acts as a repressor, typical of this family of regulators. We further defined the MSMEG_3765 regulon using genome-wide transcriptional profiling and confirmed that the MSMEG_3762/63/65 promoter is induced under acid-nitrosative stress. A putative 36 bp regulatory motif was identified upstream of the gene clusters in both M. smegmatis and M. tuberculosis and purified recombinant MSMEG_3765 protein was found to bind to DNA fragments containing this motif from both M. smegmatis and M. tuberculosis upstream regulatory regions. These results suggest that the TetR repressor MSMEG_3765/Rv1685c controls expression of an efflux pump with an, as yet, undefined role in the mycobacterial response to acid-nitrosative stress.
2017-10-24 | E-MTAB-5869 | biostudies-arrayexpress
Project description:Phagocytosis requires the activation of a plethora of mechanisms that include the activation of the actin cytoskeleton guided by the Arp2/3 complex. These are promoted by activators such as the Wiskott Aldrich Syndrome Protein (WASP) family members. In order to further understand the molecular mechanisms involved in the early events leading the phagocytosis of the pathogenic Mycobacterium tuberculosis, we set out to examine potential roles of miRNAs in phagocytosis using genome-wide expression profiling to identify miRNAs differentially regulated following mycobacterial infection. One of the miRNAs activated upon infection of mouse macrophages with the non-pathogenic Mycobacterium smegmatis, the widely conserved miR-142-3p, was predicted and confirmed to target the Neural-WASP (N-WASP). Upregulating of miR-142-3p in mouse macrophages inversely correlated with levels of N-WASP, upon infection with live pathogenic and non-pathogenic mycobacteria, suggesting an active role of Mycobacterium tuberculosis on the regulation of phagocytosis, at the post-transcriptional level, in host cells. The reduction of N-WASP correlated with a reduced internalization of bacteria per macrophage, independently of the phagocytosis index. Furthermore, the downregulation of WASP levels accompanied those of N-WASP, at early but not at late time points, suggesting a closely regulatory mechanism among both family members, dependent on the time frame of the phagocytosis. Additionally, upregulating of miR-142-3p promoted the change in the protein levels of another predicted and confirmed target, the Cofilin2 protein, in a phagocytosis-independent fashion. Downregulation experiments promoted aberrant morphologic phenotypes in macrophages, similar to observed by others in PBMCs of humans with Wiskott Aldrich Syndrome, suggesting the strong involvement of miR-142-3p on the regulation of the actin machinery in macrophages. Altogether these results show for the first time that miRNAs are involved in the regulation of actin-mediated phagocytosis of pathogenic bacteria and that these are direct targets of Mycobacterium tuberculosis. Expression analysis of mouse macrophage cell line J774A.1 in response to infection with Mycobacterium smegmatis mc2155 EGFP. Three biological replicates each for uninfected and infected samples.
2013-06-16 | E-GEOD-23429 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE36341: mRNA degradation in Mycobacterium tuberculosis under aerobic conditions GSE36342: mRNA degradation in Mycobacterium smegmatis under aerobic conditions GSE36343: mRNA degradation in Mycobacterium tuberculosis during cold and hypoxic stress GSE36344: mRNA degradation in Mycobacterium tuberculosis with DosR ectopically induced Refer to individual Series
2012-03-08 | E-GEOD-36345 | biostudies-arrayexpress
Project description:Purpose: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. Experimental design: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. Results: Twelve soluble proteins from Mtb, including one protein complex, were purified to homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and mass spectrometry. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. Conclusion and clinical relevance: The established workflow comprises a simple, cost-effective and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.
2018-06-27 | PXD004133 | Pride
Project description:The innate immune system provides the first response to pathogen infection and orchestrates the activation of the adaptive immune system. Though a large component of the innate immune response is common to all infections, pathogen-specific innate immune responses have been documented as well. The innate immune response is thought to be especially critical for fighting infection with Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB). While TB can be a deadly disease, only 5-10% of individuals infected with MTB develop active disease, and this inter-individual variation is, at least partly, heritable. Studies of inter-individual variation in the innate immune response to MTB infection may therefore shed light on the genetic basis for variation in susceptibility to TB. Yet, to date, we still do not know which properties of the innate immune response are specific to MTB infection and which represent a general response to pathogen infection. To begin addressing this gap, we infected macrophages with eight different bacterial pathogens, including different MTB strains and related mycobacteria, and studied the transcriptional response to infection. We found that although the gene expression changes were largely consistent across the bacterial infection treatments, we were able to identify a novel subset of genes whose regulation was affected specifically by infection with mycobacteria. Genetic variants that are associated with regulatory differences in these genes should be considered candidate loci for explaining inter-individual susceptibility TB. RNA-seq of monocyte-derived macrophages isolated from 6 healthy European males at 4, 18, and 48 hours post-infection with the following 8 bacteria: Mycobacterium tuberculosis (MTB) H37Rv, Mycobacterium tuberculosis GC1237, MTB GC1237, bacillus Calmette-Guérin (BCG), Mycobacterium smegmatis, Yersinia pseudotuberculosis, Salmonella typhimurium, and Staphylococcus epidermidis. table-s1.txt is a tab-delimited text file that contains the batch-corrected log2 counts per million for each of the 156 samples, as well as the Ensembl gene ID and gene name. BCG = bacillus Calmette-Guérin GC = Mycobacterium tuberculosis GC1237 Rv = Mycobacterium tuberculosis (MTB) H37Rv Rv+ = heat-inactivated MTB H37Rv Salm = Salmonella typhimurium Smeg = Mycobacterium smegmatis Staph = Staphylococcus epidermidis Yers = Yersinia pseudotuberculosis
2015-03-31 | E-GEOD-67427 | biostudies-arrayexpress
Project description:The anti-mycobacterial activity of C17 diynes has been described previously, however, their mode of action remains unknown. Microarray techniques were used to explore the genetic regulation reponses of Mycobacterium smegmatis to treatment with the C17 diynes, falcarinol and panaxydol. Our analyses showed a distinct mode of action of the C17 diynes when compared with commonly used anti-mycobacterial drugs. In addition, geneset enrichment analysis, pathway enrichment analysis and PASS analysis revealed significant gene ontology terms, pathways and potential modes of action, respectively. Combing the results of the three analyses, we hypothesize that the C17 diynes inhibit fatty acid biosynthesis, specifically phospholipid synthesis in mycobacteira. Mycobacterium smegmatis MC2 155 was treated with 10 times of MIC90 of falcarinol, panaxydol, isoniazid, ethambutol and kanamycin for 6 hours with at least 6 independent biological replicates.
2015-02-23 | E-GEOD-64323 | biostudies-arrayexpress
Project description:Rationale: Tuberculosis has a devastating impact on global health by claiming nearly 1.4 million lives each year. During infection Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, produces heterogenous populations some of which don’t produce colonies on agar but grow in liquid media and often require supplementation with culture supernatants or recombinant Resuscitation-promoting factor, thus defined as differentially culturable bacilli. Objectives: to evaluate whether exposure to nitric oxide (NO), a well-known host defence molecule, alters mycobacterial growth phenotypes and drives generation of Rpf-dependent differentially culturable bacilli. Methods: a novel NO donor was synthesised and tested against Mtb and Mycobacterium bovis BCG in vitro, followed by growth assays, flow cytometry analysis and assessment of transcriptomic responses. Resuscitation-promoting factor (Rpf) inhibitors were used to characterise the role of Rpf proteins in the reactivation of NO-treated mycobacteria. Mycobacterial phenotypes were also investigated during infection of THP-1 macrophages activated with retinoic acid and vitamin D3. Measurements and Main Results: differentially culturable mycobacteria were generated after exposure to the novel NO donor or during infection of activated THP-1 cells. Resuscitation of these differentially culturable bacilli was largely abolished by specific Rpf inhibitors. Transcriptomic analysis revealed redox-associated stress signatures mediated by SigH and SigF, with significant down-regulation of ribosome and cell wall architecture genes, including rpfA, rpfB and rpfE, and induction of genes involved in response to thiol stress, sulphur metabolism and iron acquisition. Conclusion: Our study provides mechanistic insights into the generation of Rpf-dependent Mtb during tuberculosis and outlines a critical role of NO in this process.
2023-09-01 | E-MTAB-10776 | biostudies-arrayexpress
Project description:Analysis of Metfromin induced changes in the lung cells of Mycobacterium tuberculosis infected mouse at gene expression level. The hypothesis tested in the present study was whether metformin has any effect on the host immune response in Mycobacterium tuberculosis infected mice? Results provide important information on the effect of metformin on the inflammatory response and immune activation associated with mycobacterial infection. In conclusion, Metfromin normalizes the chronic inflammation associated with Mycobacterium tuberculosis infection.
2014-12-02 | GSE57275 | GEO
Project description:Mycobacterium smegmatis SigF is a group III sigma factor. Its ortholog in M. tuberculosis is reported to have role in regulation and function of cell wall components. In present study we have created an M. smegmatis ΔsigF mutant by allele exchange method. M. smegmatis sigF mutant shows non pigmented phenotype and is more sensitive to hydrogen peroxide generated oxidative stress. DNA microarray analysis of M. smegmatis wild type and ΔsigF mutant suggests that SigF in this species controls the expression of several energy and central intermediary metabolism genes along with regulation of carotenoid biosynthesis.
2010-08-20 | GSE19774 | GEO
Project description:Mycobacterium smegmatis encodes three ESX secretion systems, ESX-1, -3 and -4. Previous research has suggested localisation of the ESX-1 secretion system at the growing polar region of M. smegmatis. In a seperate study, we demonstrated unipolar localisation of EccA3 from ESX-3 at the growing polar region of M. smegmatis. Here we demonstrate that affinity purification of N-terminally FLAG-tagged MSMEG_0615 (EccA3) followed by mass spectrometry resulted in the co-purification proteins previously identified in the membrane fractions of M. tuberculosis. Furthermore, we demonstrate that EccA3 co-purified with proteins known to localise at the growing polar region of asymmetrically growing M. smegmatis, including Wag31 and CwsA.
2022-06-09 | PXD018978 | Pride