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Pagán-Ramos, E., J. Song, M. McFalone, M. H. Mudd i V. Deretic. "Oxidative Stress Response and Characterization of theoxyR-ahpC and furA-katG Loci inMycobacterium marinum". Journal of Bacteriology 180, nr 18 (15.09.1998): 4856–64. http://dx.doi.org/10.1128/jb.180.18.4856-4864.1998.
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ABSTRACT Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and likeMycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate geneahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to theoxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region ofahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katGexpression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream ofkatG in M. marinum. The furA-katGlinkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC andkatG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.
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Hestvik, Anne Lise K., Zakaria Hmama i Yossef Av-Gay. "Kinome Analysis of Host Response to Mycobacterial Infection: a Novel Technique in Proteomics". Infection and Immunity 71, nr 10 (październik 2003): 5514–22. http://dx.doi.org/10.1128/iai.71.10.5514-5522.2003.
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ABSTRACT An array of mammalian phospho-specific antibodies was used to screen for a host response upon mycobacterial infection, reflected as changes in host protein phosphorylation. Changes in the phosphorylation state of 31 known signaling molecules were tracked after infection with live or heat killed Mycobacterium bovis BCG or after incubation with the mycobacterial cell wall component lipoarabinomannan (LAM). Mycobacterial infection triggers a signaling cascade leading to activation of stress-activated protein kinase and its subsequent downstream target, c-Jun. Mycobacteria were also shown to inhibit the activation of protein kinase C ε and to induce phosphorylation of proteins not yet known to be involved in mycobacterial infection, such as the cytoskeletal protein α-adducin, glycogen synthase kinase 3β, and a receptor subunit involved in regulation of intracellular Ca2+ levels. The mycobacterial cell wall component LAM has been identified as a trigger for some of these modulation events.
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Gupta, Kuldeepkumar Ramnaresh, Gunjan Arora, Abid Mattoo i Andaleeb Sajid. "Stringent Response in Mycobacteria: From Biology to Therapeutic Potential". Pathogens 10, nr 11 (1.11.2021): 1417. http://dx.doi.org/10.3390/pathogens10111417.
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Mycobacterium tuberculosis is a human pathogen that can thrive inside the host immune cells for several years and cause tuberculosis. This is due to the propensity of M. tuberculosis to synthesize a sturdy cell wall, shift metabolism and growth, secrete virulence factors to manipulate host immunity, and exhibit stringent response. These attributes help M. tuberculosis to manage the host response, and successfully establish and maintain an infection even under nutrient-deprived stress conditions for years. In this review, we will discuss the importance of mycobacterial stringent response under different stress conditions. The stringent response is mediated through small signaling molecules called alarmones “(pp)pGpp”. The synthesis and degradation of these alarmones in mycobacteria are mediated by Rel protein, which is both (p)ppGpp synthetase and hydrolase. Rel is important for all central dogma processes—DNA replication, transcription, and translation—in addition to regulating virulence, drug resistance, and biofilm formation. Rel also plays an important role in the latent infection of M. tuberculosis. Here, we have discussed the literature on alarmones and Rel proteins in mycobacteria and highlight that (p)ppGpp-analogs and Rel inhibitors could be designed and used as antimycobacterial compounds against M. tuberculosis and non-tuberculous mycobacterial infections.
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Gerrick, Elias R., Thibault Barbier, Michael R. Chase, Raylin Xu, Josie François, Vincent H. Lin, Matthew J. Szucs i in. "Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response". Proceedings of the National Academy of Sciences 115, nr 25 (5.06.2018): 6464–69. http://dx.doi.org/10.1073/pnas.1718003115.
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One key to the success of Mycobacterium tuberculosis as a pathogen is its ability to reside in the hostile environment of the human macrophage. Bacteria adapt to stress through a variety of mechanisms, including the use of small regulatory RNAs (sRNAs), which posttranscriptionally regulate bacterial gene expression. However, very little is currently known about mycobacterial sRNA-mediated riboregulation. To date, mycobacterial sRNA discovery has been performed primarily in log-phase growth, and no direct interaction between any mycobacterial sRNA and its targets has been validated. Here, we performed large-scale sRNA discovery and expression profiling in M. tuberculosis during exposure to five pathogenically relevant stresses. From these data, we identified a subset of sRNAs that are highly induced in multiple stress conditions. We focused on one of these sRNAs, ncRv11846, here renamed mycobacterial regulatory sRNA in iron (MrsI). We characterized the regulon of MrsI and showed in mycobacteria that it regulates one of its targets, bfrA, through a direct binding interaction. MrsI mediates an iron-sparing response that is required for optimal survival of M. tuberculosis under iron-limiting conditions. However, MrsI is induced by multiple host-like stressors, which appear to trigger MrsI as part of an anticipatory response to impending iron deprivation in the macrophage environment.
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Rieder, Ronald J., Zhihui Zhao i Boris Zavizion. "New Approach for Drug Susceptibility Testing: Monitoring the Stress Response of Mycobacteria". Antimicrobial Agents and Chemotherapy 53, nr 11 (24.08.2009): 4598–603. http://dx.doi.org/10.1128/aac.00643-09.
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ABSTRACT Methods currently used for in vitro drug susceptibility testing are based on the assessment of bacterial growth-related processes. This reliance on cellular reproduction leads to prolonged incubation times, particularly for slowly growing organisms such as mycobacteria. A new rapid phenotypic method for the drug susceptibility testing of mycobacteria is described. The method is based on the detection of the physiological stress developed by susceptible mycobacterial cells in the presence of an antimicrobial compound. The induced stress was quantified by differential monitoring of the dielectric properties of the bacterial suspension, an easily measurable electronic property. The data presented here characterize the stress developed by Mycobacterium tuberculosis cells treated with rifampin (rifampicin), isoniazid, ethambutol, and pyrazinamide. Changes in the dielectric-based profiles of the drug-treated bacteria revealed the respective susceptibilities in near real time, and the susceptibilities were well correlated with conventional susceptibility test data.
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Grigorov, Artem S., Yulia V. Skvortsova, Oksana S. Bychenko, Leonid V. Aseev, Ludmila S. Koledinskaya, Irina V. Boni i Tatyana L. Azhikina. "Dynamic Transcriptional Landscape of Mycobacterium smegmatis under Cold Stress". International Journal of Molecular Sciences 24, nr 16 (11.08.2023): 12706. http://dx.doi.org/10.3390/ijms241612706.
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Bacterial adaptation to cold stress requires wide transcriptional reprogramming. However, the knowledge of molecular mechanisms underlying the cold stress response of mycobacteria is limited. We conducted comparative transcriptomic analysis of Mycobacterium smegmatis subjected to cold shock. The growth of M. smegmatis cultivated at 37oC was arrested just after exposure to cold (acclimation phase) but later (by 24 h) was resumed at a much slower rate (adaptation phase). Transcriptomic analyses revealed distinct gene expression patterns corresponding to the two phases. During the acclimation phase, differential expression was observed for genes associated with cell wall remodeling, starvation response, and osmotic pressure stress, in parallel with global changes in the expression of transcription factors and the downregulation of ribosomal genes, suggesting an energy-saving strategy to support survival. At the adaptation phase, the expression profiles were recovered, indicating restoration of the processes repressed earlier. Comparison of transcriptional responses in M. smegmatis with those in other bacteria revealed unique adaptation strategies developed by mycobacteria. Our findings shed light on the molecular mechanisms underlying M. smegmatis survival under cold stress. Further research should clarify whether the discovered transcriptional mechanisms exist in other mycobacterial species, including pathogenic Mycobacterium tuberculosis, which could be important for transmission control.
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Ung, Korine S. E., i Yossef Av-Gay. "Mycothiol-dependent mycobacterial response to oxidative stress". FEBS Letters 580, nr 11 (21.04.2006): 2712–16. http://dx.doi.org/10.1016/j.febslet.2006.04.026.
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Williams, Diana L., Tana L. Pittman, Mike Deshotel, Sandra Oby-Robinson, Issar Smith i Robert Husson. "Molecular Basis of the Defective Heat Stress Response in Mycobacterium leprae". Journal of Bacteriology 189, nr 24 (12.10.2007): 8818–27. http://dx.doi.org/10.1128/jb.00601-07.
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ABSTRACT Mycobacterium leprae, a major human pathogen, grows poorly at 37°C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.
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Saviola, Beatrice, Samuel C. Woolwine i William R. Bishai. "Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology". Infection and Immunity 71, nr 3 (marzec 2003): 1379–88. http://dx.doi.org/10.1128/iai.71.3.1379-1388.2003.
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ABSTRACT A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.
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Fernandes, Norvin D., Qi-long Wu, Dequan Kong, Xiaoling Puyang, Sumeet Garg i Robert N. Husson. "A Mycobacterial Extracytoplasmic Sigma Factor Involved in Survival following Heat Shock and Oxidative Stress". Journal of Bacteriology 181, nr 14 (15.07.1999): 4266–74. http://dx.doi.org/10.1128/jb.181.14.4266-4274.1999.
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ABSTRACT Extracytoplasmic function (ECF) sigma factors are a heterogeneous group of alternative sigma factors that regulate gene expression in response to a variety of conditions, including stress. We previously characterized a mycobacterial ECF sigma factor, SigE, that contributes to survival following several distinct stresses. A gene encoding a closely related sigma factor, sigH, was cloned fromMycobacterium tuberculosis and Mycobacterium smegmatis. A single copy of this gene is present in these and other fast- and slow-growing mycobacteria, including M. fortuitum and M. avium. While the M. tuberculosis and M. smegmatis sigH genes encode highly similar proteins, there are multiple differences in adjacent genes. The single in vivo transcriptional start site identified inM. smegmatis and one of two identified in M. bovis BCG were found to have −35 promoter sequences that match the ECF-dependent −35 promoter consensus. Expression from these promoters was strongly induced by 50°C heat shock. In comparison to the wild type, an M. smegmatis sigH mutant was found to be more susceptible to cumene hydroperoxide stress but to be similar in logarithmic growth, stationary-phase survival, and survival following several other stresses. Survival of an M. smegmatis sigH sigE double mutant was found to be markedly decreased following 53°C heat shock and following exposure to cumene hydroperoxide. Expression of the second gene in the sigH operon is required for complementation of the sigH stress phenotypes. SigH is an alternative sigma factor that plays a role in the mycobacterial stress response.
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Rybniker, Jan, Florence Pojer, Jan Marienhagen, Gaëlle S. Kolly, Jeffrey M. Chen, Edeltraud van Gumpel, Pia Hartmann i Stewart T. Cole. "The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron–sulfur cluster biogenesis and oxidative stress defence". Biochemical Journal 459, nr 3 (11.04.2014): 467–78. http://dx.doi.org/10.1042/bj20130732.
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IscS of Mycobacterium tuberculosis is an essential component of iron–sulfur cluster assembly conferring resistance to oxidative stress. The strongly altered surface structure and the extensive protein-interaction network identified in the present study mirrors adaptations made in response to a heavily depleted mycobacterial ISC operon.
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Rasi, Valerio, Mei Xia, David Wood, Christopher Eickhoff i Daniel F. Hoft. "Mechanistic investigations of Granzyme A-mediated TB protective effects". Journal of Immunology 206, nr 1_Supplement (1.05.2021): 110.22. http://dx.doi.org/10.4049/jimmunol.206.supp.110.22.
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Abstract One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb), the pathogen that causes Tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. To elucidate the mechanism(s) involved in GzmA-mediated mycobacterial pathogen inhibition using a relevant in vitro human system. This GzmA was utilized to treat mycobacteria-infected primary monocytes from multiple volunteers, and their cell lysates were analyzed using 2D-DIGE global proteomics. A catalytically inactive GzmA variant was produced to study the enzymatic role in the Mycobacterial Growth Inhibition Assay (MGIA). In parallel, confocal microscopy experiments looked at GzmA internalization. We have evidence that Granzyme A’s intact active site is not necessary to inhibit the intracellular replication of mycobacteria as the S195A variant inhibits mycobacterial infection in MGIA as Wild Type (WT) GzmA. The proteomic approach identified two pathways: ER-stress response and purinergic channel receptor activation are important for GzmA-mediated intracellular mycobacterial inhibition. These results were validated in separate volunteers by western blot. Confocal microscopy experiments show that GzmA is internalized within the infected cell and clears the infection. Modulation of key proteins in the discovered pathways are being targeted to confirm our proteomic results and to test potential novel therapies that emulate GzmA. It will also be investigated if GzmA co-localizes in these cellular compartments. These studies will lead to a better understanding of GzmA biology during infection and to the development of novel host-directed therapeutics for control of Mtb infection.
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Lin, Wenwei, Paola Florez de Sessions, Garrett Hor Keong Teoh, Ahmad Naim Nazri Mohamed, Yuan O. Zhu, Vanessa Hui Qi Koh, Michelle Lay Teng Ang, Peter C. Dedon, Martin Lloyd Hibberd i Sylvie Alonso. "Transcriptional Profiling of Mycobacterium tuberculosis Exposed toIn VitroLysosomal Stress". Infection and Immunity 84, nr 9 (20.06.2016): 2505–23. http://dx.doi.org/10.1128/iai.00072-16.
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Increasing experimental evidence supports the idea thatMycobacterium tuberculosishas evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge ofM. tuberculosisresponse to the hostile lysosomal environment, we profiled the global transcriptional activity ofM. tuberculosiswhen exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication,M. tuberculosisexpressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified theglgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection andin vitrostress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding geneRv1258cwas selected for validation. AnM. tuberculosis ΔRv1258cmutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.
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Yimcharoen, Manita, Sukanya Saikaew, Usanee Wattananandkul, Ponrut Phunpae, Sorasak Intorasoot, Chatchai Tayapiwatana i Bordin Butr-Indr. "Mycobacterium tuberculosis Adaptation in Response to Isoniazid Treatment in a Multi-Stress System That Mimics the Host Environment". Antibiotics 12, nr 5 (5.05.2023): 852. http://dx.doi.org/10.3390/antibiotics12050852.
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Isoniazid (INH) is an antibiotic that is widely used to treat tuberculosis (TB). Adaptation to environmental stress is a survival strategy for Mycobacterium tuberculosis and is associated with antibiotic resistance development. Here, mycobacterial adaptation following INH treatment was studied using a multi-stress system (MS), which mimics host-derived stress. Mtb H37Rv (drug-susceptible), mono-isoniazid resistant (INH-R), mono-rifampicin resistant (RIF-R), and multidrug-resistant (MDR) strains were cultivated in the MS with or without INH. The expression of stress-response genes (hspX, tgs1, icl1, and sigE) and lipoarabinomannan (LAM)-related genes (pimB, mptA, mptC, dprE1, dprE2, and embC), which play important roles in the host–pathogen interaction, were measured using real-time PCR. The different adaptations of the drug-resistant (DR) and drug-susceptible (DS) strains were presented in this work. icl1 and dprE1 were up-regulated in the DR strains in the MS, implying their roles as markers of virulence and potential drug targets. In the presence of INH, hspX, tgs1, and sigE were up-regulated in the INH-R and RIF-R strains, while icl1 and LAM-related genes were up-regulated in the H37Rv strain. This study demonstrates the complexity of mycobacterial adaptation through stress response regulation and LAM expression in response to INH under the MS, which could potentially be applied for TB treatment and monitoring in the future.
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Pagán-Ramos, Eileen, Sharon S. Master, Christopher L. Pritchett, Renate Reimschuessel, Michele Trucksis, Graham S. Timmins i Vojo Deretic. "Molecular and Physiological Effects of Mycobacterial oxyR Inactivation". Journal of Bacteriology 188, nr 7 (1.04.2006): 2674–80. http://dx.doi.org/10.1128/jb.188.7.2674-2680.2006.
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ABSTRACT The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.
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Sieniawska, Elwira, Rafał Sawicki, Joanna Golus i Milen I. Georgiev. "Untargetted Metabolomic Exploration of the Mycobacterium tuberculosis Stress Response to Cinnamon Essential Oil". Biomolecules 10, nr 3 (26.02.2020): 357. http://dx.doi.org/10.3390/biom10030357.
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The antimycobacterial activity of cinnamaldehyde has already been proven for laboratory strains and for clinical isolates. What is more, cinnamaldehyde was shown to threaten the mycobacterial plasma membrane integrity and to activate the stress response system. Following promising applications of metabolomics in drug discovery and development we aimed to explore the mycobacteria response to cinnamaldehyde within cinnamon essential oil treatment by untargeted liquid chromatography–mass spectrometry. The use of predictive metabolite pathway analysis and description of produced lipids enabled the evaluation of the stress symptoms shown by bacteria. This study suggests that bacteria exposed to cinnamaldehyde could reorganize their outer membrane as a physical barrier against stress factors. They probably lowered cell wall permeability and inner membrane fluidity, and possibly redirected carbon flow to store energy in triacylglycerols. Being a reactive compound, cinnamaldehyde may also contribute to disturbances in bacteria redox homeostasis and detoxification mechanisms.
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Boshoff, Helena I., Valerie Mizrahi i Clifton E. Barry. "Effects of Pyrazinamide on Fatty Acid Synthesis by Whole Mycobacterial Cells and Purified Fatty Acid Synthase I". Journal of Bacteriology 184, nr 8 (15.04.2002): 2167–72. http://dx.doi.org/10.1128/jb.184.8.2167-2172.2002.
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ABSTRACT The effects of low extracellular pH and intracellular accumulation of weak organic acids were compared with respect to fatty acid synthesis by whole cells of Mycobacterium tuberculosis and Mycobacterium smegmatis. The profile of fatty acids synthesized during exposure to benzoic, nicotinic, or pyrazinoic acids, as well as that observed during intracellular hydrolysis of the corresponding amides, was not a direct consequence of modulation of fatty acid synthesis by these compounds but reflected the response to inorganic acid stress. Analysis of fatty acid synthesis in crude mycobacterial cell extracts demonstrated that pyrazinoic acid failed to directly modulate the fatty acid synthase activity catalyzed by fatty acid synthase I (FAS-I). However, fatty acid synthesis was irreversibly inhibited by 5-chloro-pyrazinamide in a time-dependent fashion. Moreover, we demonstrate that pyrazinoic acid does not inhibit purified mycobacterial FAS-I, suggesting that this enzyme is not the immediate target of pyrazinamide.
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Baros, Seanantha S., Jonathan M. Blackburn i Nelson C. Soares. "Phosphoproteomic Approaches to Discover Novel Substrates of Mycobacterial Ser/Thr Protein Kinases". Molecular & Cellular Proteomics 19, nr 2 (15.12.2019): 233–44. http://dx.doi.org/10.1074/mcp.r119.001668.
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Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kinases. In addition, we assess substrate specificity patterns across different mycobacterial STPKs by analyzing reported sets of phosphopeptides, in order to determine whether novel motifs or consensus regions exist for mycobacterial Ser/Thr phosphorylation sites. This review focuses on MS-based techniques employed for STPK substrate identification in mycobacteria, while highlighting the advantages and challenges of the various applications.
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Man, DeDe Kwun-Wai, Tokuwa Kanno, Giorgia Manzo, Brian D. Robertson, Jenny K. W. Lam i A. James Mason. "Rifampin- or Capreomycin-Induced Remodeling of the Mycobacterium smegmatis Mycolic Acid Layer Is Mitigated in Synergistic Combinations with Cationic Antimicrobial Peptides". mSphere 3, nr 4 (18.07.2018): e00218-18. http://dx.doi.org/10.1128/msphere.00218-18.
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ABSTRACT The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here, we investigate the response of Mycobacterium smegmatis to the presence of rifampin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs that are distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) metabolomics, diphenylhexatriene (DPH) fluorescence anisotropy measurements, and laurdan emission spectroscopy, we show that M. smegmatis responds to challenge with rifampin or capreomycin by substantially altering its metabolism and, in particular, by remodeling the cell envelope. Overall, the changes are consistent with a reduction of trehalose dimycolate and an increase of trehalose monomycolate and are associated with increased rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes, respectively, in mycomembrane metabolites and did not induce a significant increase in the rigidity of the mycolic acid layer. Furthermore, the response to rifampin or capreomycin was significantly reduced when these were combined with D-LAK120-HP13 and D-LAK120-A, respectively, suggesting a possible mechanism for the synergy of these combinations. The remodeling of the mycomembrane in M. smegmatis is therefore identified as an important countermeasure deployed against rifampin or capreomycin, but this can be mitigated and the efficacy of rifampin or capreomycin potentiated by combining the drug with AMPs. IMPORTANCE We have used a combined NMR metabolomics/biophysical approach to better understand differences in the mechanisms of two closely related antimicrobial peptides, as well as the response of the model organism Mycobacterium smegmatis to challenge with first- or second-line antibiotics used against mycobacterial pathogens. We show that, in addition to membrane damage, the triggering of oxidative stress may be an important part of the mechanism of action of one AMP. The metabolic shift that accompanied rifampin and, particularly, capreomycin challenge was associated with modest and more dramatic changes, respectively, in the mycomembrane, providing a rationale for how the response to one antibiotic may affect bacterial penetration and, hence, the action of another. This study presents the first insights into how antimicrobial peptides may operate synergistically with existing antibiotics whose efficacy is waning or sensitize MDR mycobacteria and/or latent mycobacterial infections to them, prolonging the useful life of these drugs.
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Dussurget, Olivier, i Issar Smith. "Interdependence of mycobacterial iron regulation, oxidative-stress response and isoniazid resistance". Trends in Microbiology 6, nr 9 (wrzesień 1998): 354–58. http://dx.doi.org/10.1016/s0966-842x(98)01307-9.
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Provvedi, Roberta, Francesca Boldrin, Francesco Falciani, Giorgio Palù i Riccardo Manganelli. "Global transcriptional response to vancomycin in Mycobacterium tuberculosis". Microbiology 155, nr 4 (1.04.2009): 1093–102. http://dx.doi.org/10.1099/mic.0.024802-0.
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In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin–antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor σ E confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response.
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Wu, Chia-wei, Shelly K. Schmoller, Sung Jae Shin i Adel M. Talaat. "Defining the Stressome of Mycobacterium avium subsp. paratuberculosis In Vitro and in Naturally Infected Cows". Journal of Bacteriology 189, nr 21 (10.08.2007): 7877–86. http://dx.doi.org/10.1128/jb.00780-07.
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ABSTRACT Mycobacterium avium subsp. paratuberculosis causes an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile of M. avium subsp. paratuberculosis exposed to different stress conditions, or shed in cow feces, could improve our understanding of the pathogenesis of M. avium subsp. paratuberculosis. In this report, the stress response of M. avium subsp. paratuberculosis on a genome-wide level (stressome) was defined for the first time using DNA microarrays. Expression data analysis revealed unique gene groups of M. avium subsp. paratuberculosis that were regulated under in vitro stressors while additional groups were regulated in the cow samples. Interestingly, acidic pH induced the regulation of a large number of genes (n = 597), suggesting the high sensitivity of M. avium subsp. paratuberculosis to acidic environments. Generally, responses to heat shock, acidity, and oxidative stress were similar in M. avium subsp. paratuberculosis and Mycobacterium tuberculosis, suggesting common pathways for mycobacterial defense against stressors. Several sigma factors (e.g., sigH and sigE) were differentially coregulated with a large number of genes depending on the type of each stressor. Subsequently, we analyzed the virulence of six M. avium subsp. paratuberculosis mutants with inactivation of differentially regulated genes using a murine model of paratuberculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g., lipN). Overall, the employed approach profiled mycobacterial genetic networks triggered by variable stressors and identified a novel set of putative virulence genes. A similar approach could be applied to analyze other intracellular pathogens.
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White, Mark J., Hongjun He, Renee M. Penoske, Sally S. Twining i Thomas C. Zahrt. "PepD Participates in the Mycobacterial Stress Response Mediated through MprAB and SigE". Journal of Bacteriology 192, nr 6 (8.01.2010): 1498–510. http://dx.doi.org/10.1128/jb.01167-09.
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ABSTRACT Currently, one-third of the world's population is believed to be latently infected with Mycobacterium tuberculosis. The mechanisms by which M. tuberculosis establishes latent infection remain largely undefined. mprAB encodes a two-component signal transduction system required by M. tuberculosis for aspects of persistent infection. MprAB regulates a large and diverse group of genetic determinants in response to membrane stress, including the extracytoplasmic function (ECF) sigma factor sigE and the HtrA-like serine protease pepD. Recent studies have demonstrated that PepD functions as both a protease and chaperone in vitro. In addition, inactivation of pepD alters the virulence of M. tuberculosis in a mouse model system of infection. Here, we demonstrate that PepD plays an important role in the stress response network of Mycobacterium mediated through MprAB and SigE. In particular, we demonstrate that the protease activity of PepD requires the PDZ domain, in addition to the catalytic serine at position 317. pepD expression initiates from at least three promoters in M. tuberculosis, including one that is regulated by SigE and is located upstream of the mprA coding sequence. Deletion of pepD or mprAB in Mycobacterium smegmatis and M. tuberculosis alters the stress response phenotypes of these strains, including increasing sensitivity to SDS and cell wall antibiotics and upregulating the expression of stress-responsive determinants, including sigE. Taking these data together, we hypothesize that PepD utilizes its PDZ domain to recognize and process misfolded proteins at the cell membrane, leading to activation of the MprAB and SigE signaling pathways and subsequent establishment of a positive feedback loop that facilitates bacterial adaptation.
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Lu, Liang-dong, Qing Sun, Xiao-yong Fan, Yi Zhong, Yu-feng Yao i Guo-Ping Zhao. "Mycobacterial MazG Is a Novel NTP Pyrophosphohydrolase Involved in Oxidative Stress Response". Journal of Biological Chemistry 285, nr 36 (7.06.2010): 28076–85. http://dx.doi.org/10.1074/jbc.m109.088872.
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Joseph, Sunil V., G. K. Madhavilatha, R. Ajay Kumar i Sathish Mundayoor. "Comparative Analysis of Mycobacterial Truncated Hemoglobin Promoters and thegroEL2Promoter in Free-Living and Intracellular Mycobacteria". Applied and Environmental Microbiology 78, nr 18 (6.07.2012): 6499–506. http://dx.doi.org/10.1128/aem.01984-12.
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ABSTRACTThe success ofMycobacterium tuberculosisdepends on its ability to withstand and survive the hazardous environment inside the macrophages that are created by reactive oxygen intermediates, reactive nitrogen intermediates, severe hypoxia, low pH, and high CO2levels. Therefore, an effective detoxification system is required for the pathogen to persistin vivo. The genome ofM. tuberculosiscontains a new family of hemoproteins named truncated hemoglobin O (trHbO) and truncated hemoglobin N (trHbN), encoded by theglbOandglbNgenes, respectively, important in the survival ofM. tuberculosisin macrophages. Mycobacterial heat shock proteins are known to undergo rapid upregulation under stress conditions. The expression profiles of the promoters of these genes were studied by constructing transcriptional fusions with green fluorescent protein and monitoring the promoter activity in both free-living and intracellular milieus at different time points. WhereasglbNshowed an early response to the oxidative and nitrosative stresses tested,glbOgave a lasting response to lower concentrations of both stresses. At all time points and under all stress conditions tested,groEL2showed higher expression than both trHb promoters and expression of both promoters showed an increase while inside the macrophages. Real-time PCR analysis of trHb andgroEL2mRNAs showed an initial upregulation at 24 h postinfection. The presence of theglbOprotein imparted an increased survival toM. smegmatisin THP-1 differentiated macrophages compared to that imparted by theglbNandhsp65proteins. The comparative upregulation shown by both trHb promoters while grown inside macrophages indicates the importance of these promoters for the survival ofM. tuberculosisin the hostile environment of the host.
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Danelishvili, Lia, Martin Wu, Lowell S. Young i Luiz E. Bermudez. "Genomic Approach to Identifying the Putative Target of and Mechanisms of Resistance to Mefloquine in Mycobacteria". Antimicrobial Agents and Chemotherapy 49, nr 9 (wrzesień 2005): 3707–14. http://dx.doi.org/10.1128/aac.49.9.3707-3714.2005.
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ABSTRACT The emergence of mycobacterial resistance to multiple antimicrobials emphasizes the need for new compounds. The antimycobacterial activity of mefloquine has been recently described. Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium tuberculosis are susceptible to mefloquine in vitro, and activity was evidenced in vivo against M. avium. Attempts to obtain resistant mutants by both in vitro and in vivo selection have failed. To identify mycobacterial genes regulated in response to mefloquine, we employed DNA microarray and green fluorescent protein (GFP) promoter library techniques. Following mefloquine treatment, RNA was harvested from M. tuberculosis H37Rv, labeled with 32P, and hybridized against a DNA array. Exposure to 4× MIC resulted in a significant stress response, while exposure to a subinhibitory concentration of mefloquine triggered the expression of genes coding for enzymes involved in fatty acid synthesis, the metabolic pathway, and transport across the membrane and other proteins of unknown function. Evaluation of gene expression using an M. avium GFP promoter library exposed to subinhibitory concentrations of mefloquine revealed more than threefold upregulation of 24 genes. To complement the microarray results, we constructed an M. avium genomic library under the control of a strong sigma-70 (G13) promoter in M. smegmatis. Resistant clones were selected in 32 μg/ml of mefloquine (wild-type M. avium, M. tuberculosis, and M. smegmatis are inhibited by 8 μg/ml), and the M. avium genes associated with M. smegmatis resistant to mefloquine were sequenced. Two groups of genes were identified: one affecting membrane transport and one gene that apparently is involved in regulation of cellular replication.
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MEHLERT, ANGELA, JONATHAN LAMB i DOUGLAS YOUNG. "Analysis of stress-related proteins involved in the immune response to mycobacterial infection". Biochemical Society Transactions 16, nr 5 (1.10.1988): 721–22. http://dx.doi.org/10.1042/bst0160721.
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Rodriguez, G. Marcela, Martin I. Voskuil, Benjamin Gold, Gary K. Schoolnik i Issar Smith. "ideR, an Essential Gene in Mycobacterium tuberculosis: Role of IdeR in Iron-Dependent Gene Expression, Iron Metabolism, and Oxidative Stress Response". Infection and Immunity 70, nr 7 (lipiec 2002): 3371–81. http://dx.doi.org/10.1128/iai.70.7.3371-3381.2002.
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ABSTRACT The mycobacterial IdeR protein is a metal-dependent regulator of the DtxR (diphtheria toxin repressor) family. In the presence of iron, it binds to a specific DNA sequence in the promoter regions of the genes that it regulates, thus controlling their transcription. In this study, we provide evidence that ideR is an essential gene in Mycobacterium tuberculosis. ideR cannot normally be disrupted in this mycobacterium in the absence of a second functional copy of the gene. However, a rare ideR mutant was obtained in which the lethal effects of ideR inactivation were alleviated by a second-site suppressor mutation and which exhibited restricted iron assimilation capacity. Studies of this strain and a derivative in which IdeR expression was restored allowed us to identify phenotypic effects resulting from ideR inactivation. Using DNA microarrays, the iron-dependent transcriptional profiles of the wild-type, ideR mutant, and ideR-complemented mutant strains were analyzed, and the genes regulated by iron and IdeR were identified. These genes encode a variety of proteins, including putative transporters, proteins involved in siderophore synthesis and iron storage, members of the PE/PPE family, a membrane protein involved in virulence, transcriptional regulators, and enzymes involved in lipid metabolism.
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Pantuso, Traci, Mari Kogiso, Tsutomu Shinohara, Ruth Ann Henriksen, C. Kathleen Dorey i Yoshimi Shibata. "Mycobacteria induce the inactive form of cyclooxygenase-2 (COX-2) without inducing endoplasmic reticulum (ER) stress responses in local macrophages (Mνll]) (135.29)". Journal of Immunology 182, nr 1_Supplement (1.04.2009): 135.29. http://dx.doi.org/10.4049/jimmunol.182.supp.135.29.
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Abstract In response to pathogens, activated MØ frequently express COX-2, which mediates increased release of prostaglandin E2 (PGE2), regulating host defense and inflammatory responses. We previously demonstrated, however, that i.p. administration of HK- Mycobacterium bovis BCG or M. tuberculosis resulted within 24h in increased COX-2 formation, which was subcellularly dissociated from the nuclear envelope (NE), enzymatically inactive and mediated no PGE2 release in peritoneal MØ. In contrast, when normal peritoneal MØ isolated from mice or RAW264.7 MØ were activated for 24h in vitro by BCG, they expressed increased COX-2, which was subcellularly localized in the NE and mediated increased release of PGE2. To assess whether production of inactive COX-2 is associated with ER stress-dependent signaling, we measured selected ER stress protein levels in peritoneal MØ from C57Bl/6 mice and RAW264.7 MØ that were treated with BCG in vitro or in vivo. Literature indicates that in vivo administration of endotoxin has been shown to up-regulate ER stress responses. We found that ER stress proteins growth arrest and DNA-damage-inducible gene 153, glucose regulated protein 78, and the phosphorylation of eukaryotic initiation factor two were up-regulated in RAW264.7 MØ when stimulated with endotoxin, tunicamycin or thapsigargin. However, no detectable changes in ER stress proteins were found in BCG-treated RAW264.7 MØ, in vitro BCG-treated peritoneal MØ or in vivo BCG-treated peritoneal MØ. Our results indicate that mycobacterial activation of MØ COX-2 expression in vivo or in vitro does not involve ER stress/unfolded protein responses. (Supported by NIH HL71711, DOD DAMD 17-03-1-0004, BCCRP 06BS-04-9615)
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Samanta, Sintu, Priyanka Biswas, Arka Banerjee, Avipsa Bose, Nida Siddiqui, Subhalaxmi Nambi, Deepak Kumar Saini i Sandhya S. Visweswariah. "A universal stress protein in Mycobacterium smegmatis sequesters the cAMP-regulated lysine acyltransferase and is essential for biofilm formation". Journal of Biological Chemistry 295, nr 6 (27.12.2019): 1500–1516. http://dx.doi.org/10.1074/jbc.ra119.011373.
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Universal stress proteins (USPs) are present in many bacteria, and their expression is enhanced under various environmental stresses. We have previously identified a USP in Mycobacterium smegmatis that is a product of the msmeg_4207 gene and is a substrate for a cAMP-regulated protein lysine acyltransferase (KATms; MSMEG_5458). Here, we explored the role of this USP (USP4207) in M. smegmatis and found that its gene is present in an operon that also contains genes predicted to encode a putative tripartite tricarboxylate transporter (TTT). Transcription of the TTT-usp4207 operon was induced in the presence of citrate and tartrate, perhaps by the activity of a divergent histidine kinase-response regulator gene pair. A usp4207-deleted strain had rough colony morphology and reduced biofilm formation compared with the WT strain; however, both normal colony morphology and biofilm formation were restored in a Δusp4207Δkatms strain. We identified several proteins whose acetylation was lost in the Δkatms strain, and whose transcript levels increased in M. smegmatis biofilms along with that of USP4207, suggesting that USP4207 insulates KATms from its other substrates in the cell. We propose that USP4207 sequesters KATms from diverse substrates whose activities are down-regulated by acylation but are required for biofilm formation, thus providing a defined role for this USP in mycobacterial physiology and stress responses.
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McLEAN, I. L., J. R. ARCHER, M. I. D. CAWLEY, F. S. PEGLEY, B. L. KIDD i P. W. THOMPSON. "SPECIFIC ANTIBODY RESPONSE TO THE MYCOBACTERIAL 65 kDa STRESS PROTEIN IN ANKYLOSING SPONDYLITIS AND RHEUMATOID ARTHRITIS". Rheumatology 29, nr 6 (1990): 426–29. http://dx.doi.org/10.1093/rheumatology/29.6.426.
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Pervin, K., A. Childerstone, T. Shinnick, Y. Mizushima, R. van der Zee, A. Hasan, R. Vaughan i T. Lehner. "T cell epitope expression of mycobacterial and homologous human 65-kilodalton heat shock protein peptides in short term cell lines from patients with Behçet's disease." Journal of Immunology 151, nr 4 (15.08.1993): 2273–82. http://dx.doi.org/10.4049/jimmunol.151.4.2273.
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Abstract T cell epitopes of the 65-kDa heat shock protein (HSP) were mapped in patients with Behçet's disease (BD), by stimulating T cells with the overlapping synthetic peptides derived from the sequences of the Mycobacterium tuberculosis 65-kDa HSP. Significant lymphoproliferative responses were stimulated with four HSP peptides in BD, as compared with the related disease (recurrent oral ulcers), unrelated disease, and healthy controls (p < 0.05 to 0.005). In order to assess the relative frequency of sensitized lymphocytes by these peptides, 7353 short term cell lines were generated from the lymphocytes of patients and controls. Peptides 111-125, 154-172, and 311-325 (p < 0.001) and peptide 219-233 (p < 0.02) yielded significantly greater frequency of STCL in BD than in healthy and disease controls. All but peptide 154-172 stimulated only the CD4+ subset of T cells, although there was no evidence that reactivity to the selected peptides is restricted by DR2 to DR7 Ag. HLA-B51 is significantly associated with BD, but there was no evidence that B51 was a restricting element, when B51+ patients were compared with B51- patients with BD, and with B51+ healthy control subjects. A comparative investigation was then carried out between the corresponding mycobacterial and human HSP peptides. Similar or higher lympho-proliferative responses were stimulated by the human peptides compared with the mycobacterial peptides. These results suggest that the four peptide determinants within the 65-kDa HSP might be involved in the pathogenesis of BD. Whereas the high microbial load and associated stress proteins found in oral ulceration of BD may initiate an immune response to these conserved epitopes, expression of autoreactive T cell clones might be stimulated by immunodominant T cell epitopes of endogenous HSP which may induce immunopathologic changes.
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Sander, Peter, K. G. Papavinasasundaram, Thomas Dick, Evangelos Stavropoulos, Kerstin Ellrott, Burkhard Springer, M. Joseph Colston i Erik C. Böttger. "Mycobacterium bovis BCG recADeletion Mutant Shows Increased Susceptibility to DNA-Damaging Agents but Wild-Type Survival in a Mouse Infection Model". Infection and Immunity 69, nr 6 (1.06.2001): 3562–68. http://dx.doi.org/10.1128/iai.69.6.3562-3568.2001.
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ABSTRACT Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by theMycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovisBCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recAgenotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.
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Wong-Baeza, Isabel, Víctor Gabriel García Paredes, Mayra Silva-Miranda, Bibiana Patricia Ruiz-Sánchez, Jessica Castañeda-Casimiro, Alejandro Hernández-Solis, Raúl Cicero-Sabido, Jeanet Serafín-López, Sergio Estrada-Parra i Iris Estrada-García. "Mycobacterium tuberculosis extracellular vesicles induce the formation of granulomalike structures in an in vitro model". Journal of Immunology 202, nr 1_Supplement (1.05.2019): 190.86. http://dx.doi.org/10.4049/jimmunol.202.supp.190.86.
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Abstract Tuberculosis (TB) is an airborne disease caused by Mycobacterium tuberculosis. Only 10% of the infected individuals develop an active disease, while 90% of the infected individuals contain the mycobacteria inside structures known as granulomas, in a latent, asymptomatic state. The lung granuloma is formed by a core of infected macrophages, foam cells and epithelioid macrophages, surrounded by several lymphocyte subsets (CD4 T cells, CD8 T cells, Tγδ cells, NK cells, and B cells). M. tuberculosis releases extracellular vesicles, both spontaneously and under stress conditions; these vesicles carry components, derived from the mycobacterial cell wall, that are ligands for human Toll-like receptors (TLR) 2/6 and 4; as a result, these vesicles activate the innate immune system. To determine if M. tuberculosis extracellular vesicles induce the formation of a granuloma-like structure, we used an in vitro granuloma model, with peripheral blood mononuclear cells from individuals with latent TB (positive for the Quantiferon test). We found that M. tuberculosis extracellular vesicles induced the formation of granuloma-like structures that were similar in numbers, diameters and cellular composition (percentage of monocytes, T cells, Tγδ cells, NK cells and ILCs) to the granuloma-like structures formed in response to live M. tuberculosis H37Rv. These results suggest that M. tuberculosis extracellular vesicles participate in the cellular activation and migration processes that lead to granuloma formation in the lungs of TB patients. Supported by a grant 20190106 from Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional.
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Song, Taeksun, Simon L. Dove, Kon Ho Lee i Robert N. Husson. "RshA, an anti‐sigma factor that regulates the activity of the mycobacterial stress response sigma factor SigH". Molecular Microbiology 50, nr 3 (6.10.2003): 949–59. http://dx.doi.org/10.1046/j.1365-2958.2003.03739.x.
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Tiwari, Abhinav, Gábor Balázsi, Maria Laura Gennaro i Oleg A. Igoshin. "The interplay of multiple feedback loops with post-translational kinetics results in bistability of mycobacterial stress response". Physical Biology 7, nr 3 (23.08.2010): 036005. http://dx.doi.org/10.1088/1478-3975/7/3/036005.
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Fu, Li M., i Shu C. Tai. "The Differential Gene Expression Pattern ofMycobacterium tuberculosisin Response to Capreomycin and PA-824 versus First-Line TB Drugs Reveals Stress- and PE/PPE-Related Drug Targets". International Journal of Microbiology 2009 (2009): 1–9. http://dx.doi.org/10.1155/2009/879621.
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Tuberculosis is a leading infectious disease causing millions of deaths each year. How to eradicate mycobacterial persistence has become a central research focus for developing next-generation TB drugs. Yet, the knowledge in this area is fundamentally limited and only a few drugs, notably capreomycin and PA-824, have been shown to be active against non-replicating persistent TB bacilli. In this study, we performed a new bioinformatics analysis on microarray-based gene expression data obtained from the public domain to explore genes that were differentially induced by drugs between the group of capreomycin and PA-824 and the group of mainly the first-line TB drugs. Our study has identified 42 genes specifically induced by capreomycin and PA-824. Many of these genes are related to stress responses. In terms of the distribution of identified genes in a specific category relative to the whole genome, only the categories of PE/PPE and conserved hypotheticals have statistical significance. Six among the 42 genes identified in this study are on the list of the top 100 persistence targets selected by the TB Structural Genomics Consortium. Further biological elucidation of their roles in mycobacterial persistence is warranted.
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Collett, Geoffrey, Natalia Lopez i Pedro F. Lopez. "Effects of Disseminated Mycobacterial Infection on Age-Related Macular Degeneration". Case Reports in Ophthalmology 7, nr 3 (25.11.2016): 534–42. http://dx.doi.org/10.1159/000452968.
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Our patient, in the 7th decade of life, presented with worsening blurry vision over 3 weeks. The pertinent history included nonexudative age-related macular degeneration, recent pulmonary mycobacterial infection, and autoimmune pancreatitis. The patient had decreased visual acuity in both eyes; the remaining findings of our examination were relatively benign. The diagnosis of bilateral exudative age-related macular degeneration was aided by ocular imaging. Not only were exudative changes confirmed, but one modality suggested an underlying occult choroiditis, which presumably fueled a local inflammatory drive leading to evolution of the disease. Given the choroiditis developed in the setting of a recent Mycobacterium chelonae infection, dissemination of the organism must be considered a potential culprit. Additionally, a chronic inflammatory state perhaps played a simultaneous immunologic role. We feel the proposed pathogenic mechanism outlined sufficiently accounts for the rare event, that is, development of subacute bilateral exudative maculopathy. The patient responded well to bilateral intravitreal aflibercept injections. After 1 month, visual acuity was found to be near baseline and ocular imaging showed significant resolution of the exudative changes. An additional follow-up 3 months after confirmed similar stability. This case required thorough investigation of seemingly unrelated components within the patient’s history. We stress the importance of obtaining appropriate documentation from fellow health care teams when suspicious clinical presentations arise. During our investigation, we identified cryptic retinal lesions by way of angiography – leading us to recommend usage of such methods in complex cases. We also summarize the implemented aflibercept course and the favorable response to such treatment.
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Mehra, V., B. R. Bloom, A. C. Bajardi, C. L. Grisso, P. A. Sieling, D. Alland, J. Convit, X. D. Fan, S. W. Hunter i P. J. Brennan. "A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein." Journal of Experimental Medicine 175, nr 1 (1.01.1992): 275–84. http://dx.doi.org/10.1084/jem.175.1.275.
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Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.
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Chang, Dorothy Pei Shan, i Xue Li Guan. "Metabolic Versatility of Mycobacterium tuberculosis during Infection and Dormancy". Metabolites 11, nr 2 (2.02.2021): 88. http://dx.doi.org/10.3390/metabo11020088.
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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful intracellular pathogen with the ability to withstand harsh conditions and reside long-term within its host. In the dormant and persistent states, the bacterium tunes its metabolism and is able to resist the actions of antibiotics. One of the main strategies Mtb adopts is through its metabolic versatility—it is able to cometabolize a variety of essential nutrients and direct these nutrients simultaneously to multiple metabolic pathways to facilitate the infection of the host. Mtb further undergo extensive remodeling of its metabolic pathways in response to stress and dormancy. In recent years, advancement in systems biology and its applications have contributed substantially to a more coherent view on the intricate metabolic networks of Mtb. With a more refined appreciation of the roles of metabolism in mycobacterial infection and drug resistance, and the success of drugs targeting metabolism, there is growing interest in further development of anti-TB therapies that target metabolism, including lipid metabolism and oxidative phosphorylation. Here, we will review current knowledge revolving around the versatility of Mtb in remodeling its metabolism during infection and dormancy, with a focus on central carbon metabolism and lipid metabolism.
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Saquib, Najmuddin Mohd, Shilpa Jamwal, Mukul Kumar Midha, Hirdya Narain Verma i Venkatasamy Manivel. "Quantitative Proteomics and Lipidomics Analysis of Endoplasmic Reticulum of Macrophage Infected with Mycobacterium tuberculosis". International Journal of Proteomics 2015 (16.02.2015): 1–13. http://dx.doi.org/10.1155/2015/270438.
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Even though endoplasmic reticulum (ER) stress associated with mycobacterial infection has been well studied, the molecular basis of ER as a crucial organelle to determine the fate of Mtb is yet to be established. Here, we have studied the ability of Mtb to manipulate the ultrastructural architecture of macrophage ER and found that the ER-phenotypes associated with virulent (H37Rv) and avirulent (H37Ra) strains were different: a rough ER (RER) with the former against a smooth ER (SER) with the later. Further, the functional attributes of these changes were probed by MS-based quantitative proteomics (133 ER proteins) and lipidomics (8 phospholipids). Our omics approaches not only revealed the host pathogen cross-talk but also emphasized how precisely Mtb uses proteins and lipids in combination to give rise to characteristic ER-phenotypes. H37Ra-infected macrophages increased the cytosolic Ca2+ levels by attenuating the ATP2A2 protein and simultaneous induction of PC/PE expression to facilitate apoptosis. However, H37Rv inhibited apoptosis and further controlled the expression of EST-1 and AMRP proteins to disturb cholesterol homeostasis resulting in sustained infection. This approach offers the potential to decipher the specific roles of ER in understanding the cell biology of mycobacterial infection with special reference to the impact of host response.
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Williams, Ernest P., Jong-Hee Lee, William R. Bishai, Carlo Colantuoni i Petros C. Karakousis. "Mycobacterium tuberculosis SigF Regulates Genes Encoding Cell Wall-Associated Proteins and Directly Regulates the Transcriptional Regulatory Gene phoY1". Journal of Bacteriology 189, nr 11 (23.03.2007): 4234–42. http://dx.doi.org/10.1128/jb.00201-07.
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ABSTRACT Mycobacterium tuberculosis SigF is homologous to stress response and sporulation sigma factors in many bacteria. Consistent with a possible role in mycobacterial survival under stress conditions, M. tuberculosis sigF is strongly induced within cultured human macrophages and upon nutrient starvation, and SigF has been implicated in M. tuberculosis entry into stationary phase. On the other hand, SigF appears to contribute to the immune pathology of tuberculosis (TB), and a sigF-deficient mutant has altered cell membrane properties. Using an M. tuberculosis sigF deletion mutant, we show here that sigF is not required for bacillary survival under nutrient starvation conditions and within activated murine macrophages or for extracellular persistence in an in vivo granuloma model of latent TB infection. Using a chemically inducible recombinant strain to conditionally overexpress sigF, we did not observe arrest or retardation of growth in exponentially growing cultures or reduced susceptibility to rifampin and isoniazid. Consistent with our hypothesis that SigF may mediate TB immunopathogenesis by altering cell membrane properties, we found that overexpression of sigF resulted in the differential regulation of many cell wall-associated proteins, including members of the MmpL, PE, and PPE families, several of which have been shown to influence host-pathogen interactions. The most highly upregulated gene by quantitative reverse transcription-PCR at all time points following sigF induction was Rv3301c (phoY1), which encodes a probable transcriptional regulatory protein homologous to PhoU proteins involved in regulation of phosphate uptake. Using in vitro transcription analysis, we show that SigF directly regulates phoY1, whose proposed promoter sequence is GGATTG-N16-GGGTAT.
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Sun, Sarah J., Anne Dumaine, L. Charlotte J. de Bree, Mihai G. Netea i Luis B. Barreiro. "BCG vaccination impacts the epigenetic landscape of progenitor cells in human bone marrow". Journal of Immunology 208, nr 1_Supplement (1.05.2022): 164.05. http://dx.doi.org/10.4049/jimmunol.208.supp.164.05.
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Abstract The BCG vaccine, administered to more than 4 billion people worldwide, is designed to protect against Mycobacterium tuberculosis infection. Interestingly, clinical studies suggest that BCG also provides a degree of protection against heterologous infections, implicating other facets of the immune system apart from adaptive immunity. It has been hypothesized that BCG vaccination can leave immunological “scars” within hematopoietic stem and progenitor cells (HSPCs) that further impact downstream innate immune cell function. This is supported by studies in mice demonstrating that exposure to BCG leads to expansion and differential gene expression within hematopoietic stem cells and multipotent progenitors (HSCs/MPPs). However, very little is known about the impact of BCG vaccination on human bone marrow. Here we performed droplet-based scRNA- and scATAC-sequencing on human bone marrow aspirates from 20 healthy individuals, both before and 90 days after intradermal BCG vaccination or placebo. Over 1000 sites of differential chromatin accessibility across multiple CD34 subpopulations were present 90 days following BCG vaccination. Peaks of differential chromatin accessibility were enriched for binding of lineage determining and stress transcription factors, some of which were upregulated on the gene expression level. Expression levels of a subset of BCG-induced genes were found to significantly correlate with increased Il1b secretion of donor paired PBMCs in response to a C. albicans challenge. These data indicate that BCG vaccination re-wires transcription factor activity in human bone marrow in a way that may be linked to responses of PBMCs to secondary immune challenge with non-mycobacterial pathogens.
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Pradhan, Atul, Nagaraja Mukkayyan, Kishor Jakkala i Parthasarathi Ajitkumar. "Mycobacterial Populations Partly Change the Proportions of the Cells Undergoing Asymmetric/Symmetric Divisions in Response to Glycerol Levels in Growth Medium". Cells 10, nr 5 (11.05.2021): 1160. http://dx.doi.org/10.3390/cells10051160.
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Twenty to thirty percent of the septating mycobacterial cells of the mid-log phase population showed highly deviated asymmetric constriction during division (ACD), while the remaining underwent symmetric constriction during division (SCD). The ACD produced short-sized cells (SCs) and normal/long-sized cells (NCs) as the sister–daughter cells, but with significant differential susceptibility to antibiotic/oxidative/nitrite stress. Here we report that, at 0.2% glycerol, formulated in the Middlebrook 7H9 medium, a significantly high proportion of the cells were divided by SCD. When the glycerol concentration decreased to 0.1% due to cell-growth/division, the ACD proportion gradually increased until the ACD:SCD ratio reached ~50:50. With further decrease in the glycerol levels, the SCD proportion increased with concomitant decrease in the ACD proportion. Maintenance of glycerol at 0.1%, through replenishment, held the ACD:SCD proportion at ~50:50. Transfer of the cells from one culture with a specific glycerol level to the supernatant from another culture, with a different glycerol level, made the cells change the ACD:SCD proportion to that of the culture from which the supernatant was taken. RT-qPCR data showed the possibility of diadenosine tetraphosphate phosphorylase (MSMEG_2932), phosphatidylinositol synthase (MSMEG_2933), and a Nudix family hydrolase (MSMEG_2936) involved in the ACD:SCD proportion-change in response to glycerol levels. We also discussed its physiological significance.
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Rasi, Valerio, Mei Xia, David Wood, Christopher Eickhoff i Daniel F. Hoft. "Mechanistic investigations of Granzyme A-mediated TB protective effects". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 227.10. http://dx.doi.org/10.4049/jimmunol.204.supp.227.10.
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Abstract One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA when released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen. To elucidate the mechanism(s) involved in this inhibition, the lab has purified human GzmA. We have proven that Granzyme A’s intact active site is necessary to inhibit the intracellular replication of mycobacteria. The substitution of serine to alanine in the active site rendered the protein unable to control the mycobacterial infection as measured by mycobacterial growth inhibition assays, and the measurement of the pro-inflammatory marker TNF. Conversely, the effects of highly pure and active GzmA on mycobacterial replication inside human macrophages have been investigated using systems biology. A 2D-DIGE proteomic approach using adherent human monocytes has been performed to identify the pathways stimulated by GzmA involved in control of mycobacterial replication. Our analysis indicates that ER-stress responses (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Modulation of key proteins in these pathways are currently being targeted by pharmaceutical intervention and gene alteration to test the effects on GzmA-mediated inhibitory effects. Confirmation that these pathways are involved in pathogen clearance will lead to the development of novel host-directed therapies for control of Mtb infection.
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Pawaria, Sudesh, Amrita Lama, Manoj Raje i Kanak L. Dikshit. "Responses of Mycobacterium tuberculosis Hemoglobin Promoters to In Vitro and In Vivo Growth Conditions". Applied and Environmental Microbiology 74, nr 11 (4.04.2008): 3512–22. http://dx.doi.org/10.1128/aem.02663-07.
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ABSTRACT The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H2O2, but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced ∼2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased ∼2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host.
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Genestet, Charlotte, Elisabeth Hodille, Alexia Barbry, Jean-Luc Berland, Jonathan Hoffmann, Emilie Westeel, Fabiola Bastian i in. "Rifampicin exposure reveals within-host Mycobacterium tuberculosis diversity in patients with delayed culture conversion". PLOS Pathogens 17, nr 6 (24.06.2021): e1009643. http://dx.doi.org/10.1371/journal.ppat.1009643.
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Mycobacterium tuberculosis (Mtb) genetic micro-diversity in clinical isolates may underline mycobacterial adaptation to tuberculosis (TB) infection and provide insights to anti-TB treatment response and emergence of resistance. Herein we followed within-host evolution of Mtb clinical isolates in two cohorts of TB patients, either with delayed Mtb culture conversion (> 2 months), or with fast culture conversion (< 2 months). We captured the genetic diversity of Mtb isolates obtained in each patient, by focusing on minor variants detected as unfixed single nucleotide polymorphisms (SNPs). To unmask antibiotic tolerant sub-populations, we exposed these isolates to rifampicin (RIF) prior to whole genome sequencing (WGS) analysis. Thanks to WGS, we detected at least 1 unfixed SNP within the Mtb isolates for 9/15 patients with delayed culture conversion, and non-synonymous (ns) SNPs for 8/15 patients. Furthermore, RIF exposure revealed 9 additional unfixed nsSNP from 6/15 isolates unlinked to drug resistance. By contrast, in the fast culture conversion cohort, RIF exposure only revealed 2 unfixed nsSNP from 2/20 patients. To better understand the dynamics of Mtb micro-diversity, we investigated the variant composition of a persistent Mtb clinical isolate before and after controlled stress experiments mimicking the course of TB disease. A minor variant, featuring a particular mycocerosates profile, became enriched during both RIF exposure and macrophage infection. The variant was associated with drug tolerance and intracellular persistence, consistent with the pharmacological modeling predicting increased risk of treatment failure. A thorough study of such variants not necessarily linked to canonical drug-resistance, but which are prone to promote anti-TB drug tolerance, may be crucial to prevent the subsequent emergence of resistance. Taken together, the present findings support the further exploration of Mtb micro-diversity as a promising tool to detect patients at risk of poorly responding to anti-TB treatment, ultimately allowing improved and personalized TB management.
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Wick, Cecilia, Elisabeth Onestingel, Egon Demetz, Hermann Dietrich i Georg Wick. "Oral Tolerization with Mycobacterial Heat Shock Protein 65 Reduces Chronic Experimental Atherosclerosis in Aged Mice". Gerontology 64, nr 1 (15.09.2017): 36–48. http://dx.doi.org/10.1159/000480436.
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Background: Atherosclerosis is a chronic inflammatory disease of the artery wall where both innate and adaptive immunity play important roles. Modulation of the immune response against the stress protein antigen, heat shock protein (HSP) 60, by administration of mycobacterial HSP65 (mbHSP65) orally and/or nasally shows promising therapeutic results in young animals in the sense of less severe experimental atherosclerosis; however, the case of aged animals with already established atherosclerosis has so far never been investigated. Objective: To investigate if mbHSP65 immunization would further accelerate atherosclerotic progression in aged ApoE-/- mice (18 months old) with already long-established atherosclerosis and if these mice could be orally tolerized against mbHSP65. Methods: Aged wild-type (WT) and ApoE-/- mice (65 weeks) were immunized and/or orally treated with mbHSP65 and then either kept on normal chow or changed to high-cholesterol diet (HCD). Atherosclerosis was assessed by en face analysis and the number of CD4+CD25+FoxP3+ T regulatory cells (Tregs) was assessed by flow cytometry in lymph node and spleen cells. Total cholesterol and triglyceride levels were determined. Soluble mammalian HSP60 and anti-mouse HSP60 (mHSP60) and anti-mbHSP65 antibodies were detected by enzyme-linked immunosorbent assay. Results: As expected, aged WT mice had only minor lesions in the aorta, which did not change under HCD for 14 weeks. Aged ApoE-/- mice already had large complicated plaques, which increased in size under HCD. mbHSP65 immunization led to a significant aggravation of atherosclerosis in both WT and ApoE-/- mice irrespective of the nature of their diet. This increase was accompanied by increased titers of both anti-mHSP60 and anti-mbHSP65 antibodies in the circulation. The increased plaque formation could be significantly diminished with oral mbHSP65 tolerization. An increased number of Tregs and lower or unchanged levels of cholesterol and triglycerides were associated with the reduced size of aortal lesions. Conclusion: Oral tolerization against mbHSP65 could be used both to prevent and to treat chronic atherosclerosis in aged individuals.
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Brzostek, Anna, Przemysław Płociński, Alina Minias, Aneta Ciszewska, Filip Gąsior, Jakub Pawełczyk, Bożena Dziadek, Marcin Słomka i Jarosław Dziadek. "Dissecting the RecA-(In)dependent Response to Mitomycin C in Mycobacterium tuberculosis Using Transcriptional Profiling and Proteomics Analyses". Cells 10, nr 5 (11.05.2021): 1168. http://dx.doi.org/10.3390/cells10051168.
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Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level.
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Gupta, Surbhi, i Dipankar Chatterji. "Stress Responses in Mycobacteria". IUBMB Life (International Union of Biochemistry and Molecular Biology: Life) 57, nr 3 (1.03.2005): 149–59. http://dx.doi.org/10.1080/15216540500090611.
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