Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress
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ABSTRACT:
SUBMITTER: Park H
PROVIDER: S-EPMC11022373 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:Mycobacterium avium complex (MAC), including Mycobacterium avium and Mycobacterium intracellulare (MI), accounts for a significant portion of nontuberculous mycobacterial lung disease affecting immunocompromised and lung structural disease patients. Adapting pathogens to a host-induced hostile environment is critical to establishing infection and persistence within the host. However, the cellular and molecular mechanisms of stress response for MAC still need to be elucidated. In this study, we analyzed the transcriptional profile of MI under acidic and oxidative stress conditions using RNA-seq. At the transcriptome level, 80 genes were shown [FC] ≥2.0 and p <0.05 under oxidative stress with 10 mM hydrogen peroxide. In detail, 77 genes were upregulated, while 3 genes were downregulated. Also, 878 genes were shown [FC] ≥2.0 and p <0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis revealed the activation of several pathways, including nitrogen and sulfur metabolism, under acidic stress conditions. On the contrary, oxidative stress conditions activated DNA replication and repair pathways. Our data demonstrate the activation of nitrogen and sulfur metabolism in MAC infection, which could be crucial for persistence and survival under stress conditions encountered within the host during infection. In conclusion, this study suggests the importance of stress responses in MAC pathogenesis and identifies potential therapeutic target pathways.
2024-02-21 | GSE244264 | GEO
Project description:Transcriptional profiling of M. intracellulare under acidic and oxidative stress conditions
| PRJNA1022021 | ENA
Project description:Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.
| S-EPMC3366959 | biostudies-other
Project description:BackgroundThe stress response of Saccharomyces cerevisiae has been extensively studied in the past decade. However, with the advent of recent technology in single-cell transcriptome profiling, there is a new opportunity to expand and further understanding of the yeast stress response with greater resolution on a system level. To understand transcriptomic changes in baker's yeast S. cerevisiae cells under stress conditions, we sequenced 117 yeast cells under three stress treatments (hypotonic condition, glucose starvation and amino acid starvation) using a full-length single-cell RNA-Seq method.ResultsWe found that though single cells from the same treatment showed varying degrees of uniformity, technical noise and batch effects can confound results significantly. However, upon careful selection of samples to reduce technical artifacts and account for batch-effects, we were able to capture distinct transcriptomic signatures for different stress conditions as well as putative regulatory relationships between transcription factors and target genes.ConclusionOur results show that a full-length single-cell based transcriptomic analysis of the yeast may help paint a clearer picture of how the model organism responds to stress than do bulk cell population-based methods.
| S-EPMC9960639 | biostudies-literature
Project description:Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), continues to be a public health threat. With the development and widespread of drug-resistant TB, the pressure of prevention and treatment is increasing. To determine and quantify the modified proteins, high resolution mass spectrometry were used to label and quantify the peptides and proteins modified.
2023-03-09 | PXD035802 | Pride
Project description: Not available
| S-EPMC4358006 | biostudies-literature
Project description:We have performred RNA-seq analysis of WT and ∆phoR Mtb H37Rv under normal and acidic stress, to investigate the role of PhoR in maintaning the pH homoeostasis of bacterium. RNA was extracted from exponentially growing mycobacterial cells in Middlebrook 7H9 media. For acid stress, cells at OD600 of 0.6 were pelleted, washed twice with 7H9 medium buffered with 100 mM MOPS or 2N HCl for pH 7.0 or pH 4.5, respectively, re-suspended in media of indicated pH, and finally the cells were grown further for 2 hours at 37°C. After incubation cells were 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 acidic stress conditions and mount an appropriate stress response.
2022-07-15 | GSE180161 | GEO
Project description:BackgroundMycobacterium intracellulare is a representative etiological agent of emerging pulmonary M. avium-intracellulare complex disease in the industrialized countries worldwide. The recent genome sequencing of clinical strains isolated from pulmonary M. avium-intracellulare complex disease has provided insight into the genomic characteristics of pathogenic mycobacteria, especially for M. avium; however, the genomic characteristics of M. intracellulare remain to be elucidated.ResultsIn this study, we performed comparative genomic analysis of 55 M. intracellulare and related strains such as M. paraintracellulare (MP), M. indicus pranii (MIP) and M. yonogonense. Based on the average nucleotide identity, the clinical M. intracellulare strains were phylogenetically grouped in two clusters: (1) the typical M. intracellulare (TMI) group, including ATCC13950 and virulent M.i.27 and M.i.198 that we previously reported, and (2) the MP-MIP group. The alignment of the genomic regions was mostly preserved between groups. Plasmids were identified between groups and subgroups, including a plasmid common among some strains of the M.i.27 subgroup. Several genomic regions including those encoding factors involved in lipid metabolism (e.g., fadE3, fadE33), transporters (e.g., mce3), and type VII secretion system (genes of ESX-2 system) were shown to be hypermutated in the clinical strains. M. intracellulare was shown to be pan-genomic at the species and subspecies levels. The mce genes were specific to particular subspecies, suggesting that these genes may be helpful in discriminating virulence phenotypes between subspecies.ConclusionsOur data suggest that genomic diversity among M. intracellulare, M. paraintracellulare, M. indicus pranii and M. yonogonense remains at the subspecies or genovar levels and does not reach the species level. Genetic components such as mce genes revealed by the comparative genomic analysis could be the novel focus for further insight into the mechanism of human pathogenesis for M. intracellulare and related strains.
| S-EPMC8025370 | biostudies-literature
Project description:The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.
| S-EPMC7465531 | biostudies-literature
Project description:The ability to maintain redox homeostasis is critical for Mycobacterium tuberculosis (Mtb) to survive the redox stress of the host. There are many antioxidant systems in Mtb to ensure its normal replication and survival in the host, and cysteine thiols are one of them. S-sulfenylation is one of the reversible modifications of cysteine thiols to resist oxidative stress. In the study, we investigated the total cysteine thiols modification and S-sulfenylation modification of Mtb proteome under the oxidative stress provided by hydrogen peroxide. To determine and quantify the S-sulfenylation modified proteins, high specific IodoTMT6plex reagents and high resolution mass spectrometry were used to label and quantify the peptides and proteins modified. There are significant differences for the total cysteine modification levels of 279 proteins and S-sulfenylation modification levels of 297 proteins under hydrogen peroxide stress. Functional enrichment analysis indicated that these cysteine-modified proteins were involved in the oxidation-reduction process, fatty acid biosynthetic process, stress response, protein repair, cell wall, etc. In conclusion, our study provides a view of cysteine modifications of the Mtb proteome under oxidative stress, revealing a series of proteins that may play a role in maintaining redox homeostasis. IMPORTANCE With the continuous spread of drug-resistant tuberculosis, there is an urgent need for new antituberculosis drugs with new mechanisms. The ability of Mtb to resist oxidative stress is extremely important for maintaining redox homeostasis and survival in the host. The reversible modifications of cysteine residues have a dual role of protection from irreversible damage to protein functions and regulation, which plays an important role in the redox homeostasis system. Thus, to discover cysteine modification changes in the proteome level under oxidative stress is quintessential to elucidate its antioxidant mechanism. Our results provided a list of proteins involved in the antioxidant process that potentially could be considered targets for drug discovery and vaccine development. Furthermore, it is the first study to determine and quantify the S-sulfenylation-modified proteins in Mtb, which provided better insight into the Mtb response to the host oxidative defense and enable a deeper understanding of Mtb survival strategies.
| S-EPMC10101118 | biostudies-literature