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Artykuły w czasopismach na temat "Mycobacterial Stress Response"
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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.
Rozprawy doktorskie na temat "Mycobacterial Stress Response"
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Sanyi, Raghad Hassan Hussein. "Studies into the role of two mycobacterial proteins in stress response and survival inside macrophages". Thesis, University of Leicester, 2016. http://hdl.handle.net/2381/36615.
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The success of M. tuberculosis lies in its ability to stay alive and persist in a potentially hostile environment represented by the macrophage phagosome. Hence there is a desperate need to identify the molecular mechanisms and associated proteins enabling mycobacterial survival and replication inside macrophages. Recent studies have shown that several mycobacterial proteins may play distinct roles during different stages of infection. This thesis was focused on investigation of the biological function of two mycobacterial proteins, Rv1219c and Rv3136 (PPE51 protein) during macrophage infection and stress response. A recent study has established that RaaS (Rv1219c in M. tuberculosis and BCG_1279c in M. bovis BCG) mediates mycobacterial survival in stationary phase and during mouse infection. RaaS (for regulator of antimicrobial-assisted survival) controls expression of ATP-dependent efflux pumps Bcg_1278c/1277c and DrrC and mediates survival of M. bovis BCG in growth non-permissive conditions. One of the aims of this project was investigating the role of RaaS in mycobacterial replication and persistence in macrophages. The result showed that ΔraaS mutant of M. bovis BCG was significantly impaired in initial survival in macrophages. Moreover, the mutant was extremely sensitive to H2O2 and low pH, the stress factors, which probably influence mycobacterial viability upon their uptake into macrophages. Treatment with reserpine, an inhibitor of ATP-dependent efflux pumps, prior to stress exposure had managed to improve the survival of the mutant suggesting that the impaired stress response of the raaS mutant was due to dysregulation of efflux pumps. While BCG_1279c and its orthologous in M. tuberculosis, Rv1219c, are important for survival inside macrophages and in stationary phase, published data has shown that Rv3136 is up-regulated during mycobacterial replication in macrophages. To establish the role of Rv3136 in macrophage infection and virulence, an rv3136 deletion mutant was generated in M. tuberculosis H37Rv. The mutant did not show any growth defect in laboratory medium. Although macrophage infection experiments did not link rv3136 to survival in macrophages, preliminary data from mouse infection indicated that Rv3136 could potentially play a role in survival inside the host.
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Santos, Ricardo. "Diversity, stress responses and ecological behaviour of Mycobacterium species". Thesis, Queen's University Belfast, 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709877.
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Mycobacterium is one of the oldest known bacterial genera and its species are commonly associated with human and animal disease, although most are free-living saprophytes which form part of a balanced microbial community in natural habitats such as water and soil Mycobacteria are highly distributed in the environment and in man-made water infrastructures mainly due to its robustness and ability to adapt Until now there are only scarce reports of their presence in extreme environments but no methodic and large-scale survey of these environments has been done in order to truly understand their distribution in such environments. This thesis encompasses the isolation of strains from 263 different positive samples and their identification using different techniques. The most commonly isolated species were Mycobacterium gordonae in Glacier National Park and Mycobacterium parascrofulaceum and Mycobacterium avium in Yellowstone National Park. Isolates were tested for their resistance co temperature, pH and different stressors, in an attempt to explain the remarkable resistance for most of the tested conditions, several cell characteristics were tested, including fatty acids composition, polyphosphate accumulation and catalase activity. Scanning Electron Microscopy was performed to have an insight into the genera! structure of the cells. Results emphasize the highly resilience of mycobacteria to different types of environmental stress. From the tested conditions, the subtle differences In the fatty add composition, in the catalase activity and polyphosphate accumulation, demonstrates that the formation of biofilm-like structures helps to understand cite highly resistance of the members of this genus.
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Hafneh, Nor Azian. "Investigation of molecular factors involved in mycobacterial stress responses and non-replicating persistence". Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42533.
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Mycobacterium tuberculosis is a remarkably successful human pathogen due to its ability to switch into dormancy or non-replicating persistence (NRP) phase driven by the host stress microenvironments. Identifying the panoply of genes or pathways involved in dormancy will progress our understanding on latent tuberculosis infection. Rv2660c and Rv2661c (conserved hypothetical proteins) and Rv1675c (transcriptional regulator) were implicated in mycobacterial stress response and transition to dormancy, hence their biological importance in M. tuberculosis biology was further explored. Rv2660c and rv2661c were highly upregulated in vitro starvation and in vivo infection model, however, recent high upregulation of a noncoding RNA, ncRv12659 in these models challenged the importance of these genes for NRP. A panel of single and double in-frame deletion mutants and over-expressing strains of rv2660c and rv2661c in M. tuberculosis were generated. A deletion of rv2660c and rv2661c also resulted in partial inactivation of ncRv12659 and rv2662 respectively. The deletion mutants exhibited normal growth in vitro and in mice. Furthermore, the strains showed unimpaired survival under nutrient starvation, hypoxia, oxidative and nitrosative stresses. Quantitative RT-PCR analysis revealed that neither target gene was highly expressed throughout starvation, oxidative and acidic pH stresses. Rv1675c (Cmr) is a redox sensor that regulates the DosR signalling pathway. Cmr binding to DNA was severely reduced by nitrosation of the two conserved cysteine residues. The cmr mutant displayed survival advantage during exposure to nitrosative stress. The over-expression of cmr or cmrC2A form (mutated cysteines) had a mild inhibitory effect on growth of M. tuberculosis. The over-expressing strain of cmrC2A was more resistant to hydrogen peroxide, suggesting that Cmr may also control the response to oxidative stress. Our study clarified the role of Rv2660c and Rv2661c in growth, NRP and infection, and further highlighted a novel Cmr-mediated regulatory network involved during nitrosative stress and transition to dormancy.
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O'Brien, Lyn. "An investigation of the killing of Mycobacterium tuberculosis by macrophages and the acid stress response of Mycobacterium smegmatis". Thesis, University of Leicester, 1995. http://hdl.handle.net/2381/35403.
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Attempts were made to activate human monocytes with immunomodulators and human macrophages with T-cell supernatants for in vitro antimycobacterial activity. Both these approaches failed. Alveolar macrophages from Mycobacterium bovis BCG-vaccinated guinea pigs have previously been shown to kill Mycobacterium tuberculosis in vitro. The guinea pig was therefore used to investigate macrophage antimycobacterial mechanisms. Tuberculocidal factors were found within lysosomes. Lysosomal fractions of macrophages from BCG-vaccinated guinea pigs significantly (P 0.001) killed M. tuberculosis. Tuberculocidal activity was not observed with macrophage lysosomal fractions from non-vaccinated guinea pigs. The specific activities of enzymes in macrophage homogenates were tested. None of the lysosomal enzymes had significantly different activities in macrophages from vaccinated and non-vaccinated guinea pigs. Strains of M. tuberculosis were sensitive to reactive nitrogen intermediates (RNI). However, guinea pig alveolar macrophages did not generate RNI on infection with mycobacteria. Macrophages from BCG-vaccinated guinea pigs killed M. tuberculosis in the presence of an inhibitor of RNI synthesis. Thus, no evidence was gained to suggest that RNI are responsible for the tuberculocidal activity of guinea pig macrophages. Aminoaldehydes have been shown to be toxic to M. tuberculosis. Ornithine decarboxylase (ODC) is the first enzyme in the pathway that synthesises aminoaldehydes. ODC activity was not elevated in macrophages twenty-four hours and six days after BCG vaccination. Increased ODC activity was not observed in macrophages infected with M. bovis BCG for twenty-four hours. ODC is therefore not responsible for any prolonged increase in aminoaldehyde production that may take place when macrophages are infected with mycobacteria. Mycobacterium tuberculosis was passaged once through the mouse. After passaging, the mycobacteria exhibited increased resistance to hydrogen peroxide but not RNI. This indicates that hydrogen peroxide is generated during the murine immune response to mycobacteria. Culturing Mycobacterium smegmatis at pH5.0 allowed the bacteria to survive subsequent incubation at pH3.5 better than bacteria grown solely at pH7.6. Mycobacterium smegmatis could therefore be adapted to lethal pH values by pre-exposing the bacteria to mildly acidic conditions.
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Hoar, Dominic Nelson. "Towards an understanding of the regulation of the oxidative stress responses of Mycobacterium tuberculosis". Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1444421/.
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Knowledge of the mechanisms by which Mycobacterium tuberculosis regulates its defence against oxidative stress will aid in the development of drugs and vaccines to combat the extensive morbidity and mortality caused by tuberculosis. In this thesis I have investigated the SenX3-RegX3 two component signal transduction system, one of 12 such systems in M. tuberculosis , which is essential for virulence in a mouse model and is similar to systems in other organisms that are involved in sensing oxygen. By complementing the attenuated growth of the senX3 and regX3 null mutants in mice I have shown that this phenotype is truly due to the targeted mutations in these strains. In vitro assays of oxygen stress have shown the senXS and regX3 null mutants to be less sensitive than the wild-type to superoxide and organic hydroperoxide stresses. Microarray analysis of mutants in the SenX3-RegX3 system after growth in microaerobic conditions suggested some functions for RegX3 but there was little overlap between strains. However, microarray comparison of a strain overproducing regX3 transcript demonstrated control of genes such as ahpC and cydB, indicating that this system may indeed play a role in the resistance of M. tuberculosis to oxidative stress. RegX3 was expressed and purified in E. coli and used to examine the interaction of this protein to different promoters, including that of senX3. Two further genes, which are probable one component regulators of oxidative stress in M. tuberculosis, Rv0465c and Rvl049, as judged by similarity to proteins in other organisms, were analysed through the isolation of null mutations. In vitro analysis suggests that Rvl049 is necessary for resistance to organic hydroperoxide stress, although neither the Rv0465c nor the Rvl049 null mutants were attenuated in macrophage or murine intravenous injection models of infection.
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Eckelt, Elke [Verfasser]. "FurA and FurB – the impact of two transcriptional metalloregulators on Mycobacterium avium ssp. paratuberculosis stress response and metal homeostasis / Elke Eckelt". Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2014. http://d-nb.info/1064862489/34.
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Nair, Rashmi Ravindran. "Unique Response and the Survival Mechanism of Mycobacterial Subpopulations against Oxidative and Nitrite Stress". Thesis, 2016. http://etd.iisc.ac.in/handle/2005/2856.
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Mycobacterial populations are known for the heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions. Such population heterogeneity had been observed in TB patients, in animal models, and in in vitro cultures. Also, the physiological relevance of population heterogeneity under nutrient starvation has been studied. However, the physiological significance of population heterogeneity in oxidative and nitrite stress has not been addressed yet. Our laboratory had earlier shown that a subpopulation of mycobacterial mid-log phase cultures divide by highly deviated asymmetric division, generating short cells and normal-sized/long cells. This proportion has been found to be consistent and reproducible, and has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum, which is known to have high levels of oxidative stress. The highly deviated asymmetric cell division has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, the physiological significance of the population heterogeneity generated by the highly deviated asymmetric division remained to be addressed. Therefore, in the present study, we addressed the physiological significance of the generation of population heterogeneity in terms of cell size in Mycobacterium smegmatis and Mycobacterium tuberculosis. In this regard, we explored whether the minor subpopulation of short cells generated in the population has any relevance in the response of mycobacteria to oxidative and nitrite stress for survival.
The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on the phenotypic heterogeneity in diverse bacterial systems and the physiological significance of such heterogeneity. Subsequently, an account of the phenotypic heterogeneity reported in mycobacteria is given, with examples of its significance implicated for survival under nutrient stress. Then an account of various studies on the oxidative and nitrite stress response of mycobacteria and on the genes involved in those processes are given. Further, the present study is justified by stating that so far there has not been any study to find out the physiological relevance of phenotypic heterogeneity on oxidative and nitrite
stress response in mycobacteria. Finally, the Introduction is concluded by stating that the present study investigates and reports for the first time the physiological significance of the minor subpopulation of short cells for survival under oxidative and nitrite stress conditions.
The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the separation of the short cells, their characterisation, stress response, and so on are given in great detail.
The Chapter 3 forms the first data chapter that presents results on the nature of response of Mycobacterium smegmatis and Mycobacterium tuberculosis against oxidative and nitrite stress. Here the cell size natural distribution, in terms of short cells and normal-sized/long cells in the mid-log phase population, the fractionation and enrichment of these subpopulations, differential susceptibility of the cells in the fractions to the stress conditions, the enhanced survival of the population upon mixing of these cell populations at the natural proportion, and the decreased survival upon mixing them at unnatural proportion are presented. The differential survival of the short cells and normal-sized/long cells was studied at a variety of stress concentrations for oxidative (H2O2) and nitrite (acidified sodium nitrite, pH 5), cell densities and exposure time to show the robustness of the phenomenon. Enhanced survival upon extended exposure to stress also has been documented. Essentially the data in this chapter shows that although the different sized populations show differential stress susceptibility to the stress conditions, their combined presence at the proportion that naturally exists in the mid-log phase population enhances the survival of the population, at the cost of the highly susceptible short cells for the enhanced survival of the less susceptible normal-sized/long cells, kin selection and altruism. The Chapter concludes with a discussion on the results.
The Chapter 4 delineates the mechanism of the altruistic phenomenon that results in the enhanced survival of the population at the sacrifice of the minor subpopulation of short cells. Here we present evidence that hydroxyl radical generated through Fenton reaction is responsible for the enhanced survival through the induction of the synthesis of catalase-peroxidase (KatG) for the degradation of H2O2. The free iron deficient short cells acquire more iron, which in turn becomes stoichiometrically detrimental to them due to the high levels of hydroxyl generation in the presence of H2O2. On the contrary, the free iron containing normal-sized/long cells do not acquire iron and hence the hydroxyl radical produced in the population becomes stoichiometrically beneficial to them. Thus, the deficiency of free iron which consequentially necessitates the short cells to acquire more iron becomes a maladaptive trait in the presence of H2O2 but gets co-opted in kin selection, for the survival of the normal-sized/long cells that form major proportion of the population – a phenomenon reminiscent of altruism. The Chapter concludes with a model depicting the entire phenomenon and a discussion on the results and their implications.
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Nair, Rashmi Ravindran. "Unique Response and the Survival Mechanism of Mycobacterial Subpopulations against Oxidative and Nitrite Stress". Thesis, 2016. http://etd.iisc.ernet.in/handle/2005/2856.
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Mycobacterial populations are known for the heterogeneity in terms of cell size, morphology, and metabolic status, which are believed to help the population survive under stress conditions. Such population heterogeneity had been observed in TB patients, in animal models, and in in vitro cultures. Also, the physiological relevance of population heterogeneity under nutrient starvation has been studied. However, the physiological significance of population heterogeneity in oxidative and nitrite stress has not been addressed yet. Our laboratory had earlier shown that a subpopulation of mycobacterial mid-log phase cultures divide by highly deviated asymmetric division, generating short cells and normal-sized/long cells. This proportion has been found to be consistent and reproducible, and has been found in the freshly diagnosed pulmonary tuberculosis patients’ sputum, which is known to have high levels of oxidative stress. The highly deviated asymmetric cell division has been found to be one of the mechanisms that mycobacteria use to generate cell size heterogeneity in the population. However, the physiological significance of the population heterogeneity generated by the highly deviated asymmetric division remained to be addressed. Therefore, in the present study, we addressed the physiological significance of the generation of population heterogeneity in terms of cell size in Mycobacterium smegmatis and Mycobacterium tuberculosis. In this regard, we explored whether the minor subpopulation of short cells generated in the population has any relevance in the response of mycobacteria to oxidative and nitrite stress for survival.
The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on the phenotypic heterogeneity in diverse bacterial systems and the physiological significance of such heterogeneity. Subsequently, an account of the phenotypic heterogeneity reported in mycobacteria is given, with examples of its significance implicated for survival under nutrient stress. Then an account of various studies on the oxidative and nitrite stress response of mycobacteria and on the genes involved in those processes are given. Further, the present study is justified by stating that so far there has not been any study to find out the physiological relevance of phenotypic heterogeneity on oxidative and nitrite
stress response in mycobacteria. Finally, the Introduction is concluded by stating that the present study investigates and reports for the first time the physiological significance of the minor subpopulation of short cells for survival under oxidative and nitrite stress conditions.
The Chapter 2 forms the Materials and Methods used in the present study. Here a detailed description of the methods used for the separation of the short cells, their characterisation, stress response, and so on are given in great detail.
The Chapter 3 forms the first data chapter that presents results on the nature of response of Mycobacterium smegmatis and Mycobacterium tuberculosis against oxidative and nitrite stress. Here the cell size natural distribution, in terms of short cells and normal-sized/long cells in the mid-log phase population, the fractionation and enrichment of these subpopulations, differential susceptibility of the cells in the fractions to the stress conditions, the enhanced survival of the population upon mixing of these cell populations at the natural proportion, and the decreased survival upon mixing them at unnatural proportion are presented. The differential survival of the short cells and normal-sized/long cells was studied at a variety of stress concentrations for oxidative (H2O2) and nitrite (acidified sodium nitrite, pH 5), cell densities and exposure time to show the robustness of the phenomenon. Enhanced survival upon extended exposure to stress also has been documented. Essentially the data in this chapter shows that although the different sized populations show differential stress susceptibility to the stress conditions, their combined presence at the proportion that naturally exists in the mid-log phase population enhances the survival of the population, at the cost of the highly susceptible short cells for the enhanced survival of the less susceptible normal-sized/long cells, kin selection and altruism. The Chapter concludes with a discussion on the results.
The Chapter 4 delineates the mechanism of the altruistic phenomenon that results in the enhanced survival of the population at the sacrifice of the minor subpopulation of short cells. Here we present evidence that hydroxyl radical generated through Fenton reaction is responsible for the enhanced survival through the induction of the synthesis of catalase-peroxidase (KatG) for the degradation of H2O2. The free iron deficient short cells acquire more iron, which in turn becomes stoichiometrically detrimental to them due to the high levels of hydroxyl generation in the presence of H2O2. On the contrary, the free iron containing normal-sized/long cells do not acquire iron and hence the hydroxyl radical produced in the population becomes stoichiometrically beneficial to them. Thus, the deficiency of free iron which consequentially necessitates the short cells to acquire more iron becomes a maladaptive trait in the presence of H2O2 but gets co-opted in kin selection, for the survival of the normal-sized/long cells that form major proportion of the population – a phenomenon reminiscent of altruism. The Chapter concludes with a model depicting the entire phenomenon and a discussion on the results and their implications.
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Jakkala, Kishor. "Response of Mycobacterium tuberculosis to Hypoxia and its physiological Significance - A Morphological and Molecular Level Study". Thesis, 2017. https://etd.iisc.ac.in/handle/2005/4677.
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Mycobacterium tuberculosis (Mtb) has evolved as an important clinical pathogen due to its ability to gain multidrug resistance, to enter into latency to persist there and to get reactivated from the latent infection in aged, immunocompromised persons to cause the disease. Mtb, which can pesist within the granulomatous parenchymal lesions in a nonreplicating persistent (NRP) stage called dormancy (clinically called latency), experience hypoxic stress. In order to understand the response of the bacilli to the hypoxic environment, several in vivo and in vitro model systems have been designed and used. Amongst the in vitro model systems, the one which closely resembles the hypoxic stress induced within the host wherein the bacilli experience a gradual depletion of oxygen, was found to be the Wayne’s in vitro hypoxia model. Therefore, it is considered to be the best model system to study the response of M. tuberculosis to the hypoxic stress in vitro as here as well the bacilli experience a gradual depletion of oxygen to progress from the actively growing mid‐log phase to NRP‐I and NRP‐II stages. Even though, several works have been carried out at the gene expression and proteomic levels to know the response of the bacilli using Wayne’s in vitro hypoxia model, surprisingly there is hardly any information available on the morphological and cellular level changes, which occur in response to hypoxia, and their correlation to molecular changes. Therefore, in the present study, using Wayne’s in vitro hypoxia model, this lacuna of information was addressed by studying morphological and cellular changes occurring to NRPI and NRP‐II M. tuberculosis cells at the ultrastructural level, correlating the changes with respective gene expression level changes, and finding out their physiological significance.
The Chapter 1, which forms the Introduction to the thesis, gives an extensive literature survey on all the different aspects of the research performed on mycobacterial cells under hypoxia, which are linked to the present study, and under other stress conditions such as nutrient depletion, pH and so on.
The Chapter 2 presents in detail all the materials and methods used to perform the experiments. A large number of biochemical, biophysical, and molecular level methods, such as scanning and transmission electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, dynamic light scattering, flow cytometry, fluorescence microscopy, real time RT‐PCR analysis, and others were used to perform the experiments.
The data Chapter 3 presents ultrastructural analysis of the M. tuberculosis cells exposed to hypoxic stress condition using Wayne’s in vitro hypoxia model. Among the different ultra-structural changes observed, this study focused mainly on the unique thickening of the outer layer (OL), during the progression of the bacilli from mid‐log phase to NRP‐I and then to NRPII stages. On the contrary, the NRP‐I and NRP‐II cells of the saprophytic mycobacterial species, Mycobacterium smegmatis, did not show thickening of the OL. Dynamic light scattering experiments using Zetasizer showed the NRP‐I and NRP‐II M. tuberculosis cells to be longer in size. Experiments using Zeta potential analyzer revealed high level of negative charge on the surface of the NRP‐I and NRP‐II M. tuberculosis bacilli. Ultrastructural studies, using scanning and transmission electron microscopy and atomic force microscopy revealed that the surface of the NRP‐II M. tuberculosis bacilli was extensively rough and uneven, unlike the smooth and even surface of the mid‐log phase cells. Fourier transform infrared spectroscopy of the groups present in the polysaccharides extracted from thickened outer layer were found to be highly anionic in nature. Polysaccharide‐specific calcofluor white staining showed the thickened outer layer of M. tuberculosis to be rich in polysaccharides. Transcriptome analysis of the respective genes involved in the synthesis of capsular polysaccharides showed significant upregulation in comparison to the same genes in the MLP cells, thus supporting the observed hypoxic adaptation of the thickening of the OL in NRP‐II M. tuberculosis cells.
The data Chapter 4 presents studies on the physiological significance of the thickening of the OL in terms of the permeability to the anti‐TB drug, rifampicin. Since it was necessary to choose a non‐bioactive variant of rifampicin in order to avoid the growth inhibitory antibiotic action on the cells, rifampicin conjugated to 5‐carboxyfluorescein (5‐FAMrifampicin), which showed only 2.5% bioactivity, was used for the permeability assay, instead of 14C‐rifampicin. The thickened OL of the NRP‐I and NRP‐II cells were found to significantly restrict the entry of 5‐FAM‐rifampicin into the cells. Mild bead beating of the NRP‐II cells, to remove the thickened OL without affecting the outer membrane integrity, as confirmed using transmission electron microscopy, restored the permeability of 5‐FAM‐rifampicin ascomparable to that into mid‐log phase cells. The entry of 5‐FAM‐rifampicin into the cells was monitored using flow cytometry analysis. M. tuberculosis cells, at 48 hrs. post‐release from= the NRP‐II stage, also showed restoration of the permeability of 5‐FAM‐rifampicin as comparable to that into mid‐log phase cells. These observations suggested that the recalcitrance of dormant bacilli to anti‐TB drugs might be due to the presence of thickened OL generated by the bacilli as a strategy to evade bactericidal effects.
The data chapters in the thesis are concluded with discussion of the findings presented in the respective chapter.
The thesis finally lists the highlights of the present study, followed up with an extensive
bibliography.
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Murdeshwar, Maya S. "Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis". Thesis, 2012. https://etd.iisc.ac.in/handle/2005/2499.
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The stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis.
Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided,
starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study.
Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included.
Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided.
Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis.
Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed.
Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed.
Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively.
Części książek na temat "Mycobacterial Stress Response"
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Bretl, Daniel J., i Thomas C. Zahrt. "Regulation of Envelope Stress Responses by Mycobacterium tuberculosis". W Regulation of Bacterial Virulence, 465–89. Washington, DC, USA: ASM Press, 2016. http://dx.doi.org/10.1128/9781555818524.ch24.
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Khan, Nooruddin, Gillipsie Minhas, K. Kala jyothi i Jyoti Sharma. "Cellular Stress Responses and Immunological Regulations During Mycobacterium tuberculosis Infection". W Mycobacterium Tuberculosis: Molecular Infection Biology, Pathogenesis, Diagnostics and New Interventions, 203–20. Singapore: Springer Singapore, 2019. http://dx.doi.org/10.1007/978-981-32-9413-4_12.
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Hameed, Saif, i Zeeshan Fatima. "Response of Mycobacterium tuberculosis to pH Stress: Promising Approach to Control Tuberculosis". W Pathogenicity and Drug Resistance of Human Pathogens, 81–92. Singapore: Springer Singapore, 2019. http://dx.doi.org/10.1007/978-981-32-9449-3_4.
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Syal, Kirtimaan, i Dipankar Chatterji. "Vitamin C: A Natural Inhibitor of Cell Wall Functions and Stress Response in Mycobacteria". W Advances in Experimental Medicine and Biology, 321–32. Singapore: Springer Singapore, 2018. http://dx.doi.org/10.1007/978-981-13-3065-0_22.
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Ivanyi, Juraj, Pamela M. Norton i Goro Matsuzaki. "Immune Responses to Stress Proteins in Mycobacterial Infections". W Stress Proteins in Medicine, 265–85. CRC Press, 2020. http://dx.doi.org/10.1201/9781003067474-16.
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Santos, Dilvani Oliveira, Arthur Willkomm Kazniakowski, Anna Fernandes Silva Chagas do Nascimento, Laura Brandão Martins, Sourou Credo Francisco Justus Zinsou, Rodolfo Avila i Maria Elena Samar. "An Auspicious Bacterium: How Mitochondria can be Beneficial to the Innate Immunity through Aerobic Exercises". W Mitochondrial DNA and the Immuno-inflammatory Response: New Frontiers to Control Specific Microbial Diseases, 1–21. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/9789815051698122030005.
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Mitochondria are highly relevant organelles with regard to their unique function in generating energy and contributing to metabolism within the cell. Furthermore, recent studies suggest that they might have an influence on the innate immune and inflammatory responses, thus affecting antiviral immunity (as example: Zika virus (ZIKV), hepatitis C virus (HCV), dengue virus and SARS-CoV-2 virus) and antibacterial immunity as well (Streptococcus pneumoniae, Mycobacterium leprae and Mycobacterium tuberculosis). Therefore, this chapter aims at bringing a relevant debate about the role of mitochondria and their multifunctional capacity. We intend to discuss the complexity of mitochondrial metabolism, especially during aerobic physical exercises, which causes the modulation of the gene expression of proteins that lead to mitochondrial proliferation and, thus, promote health. In addition, considering the injuries caused by hypoxia, this chapter also stresses the enormous potential of mitochondria to enable the survival of eukaryotic cells by allowing them to turn to aerobic respiration, as shown in previous scientific studies. In conclusion, this chapter points out the importance of mitochondrial biogenesis (both natural and stimulated biogenesis by aerobic exercise) and the benefits this organelle brings to the health, arguing that they go far beyond cellular respiration and oxidative phosphorylation.
Streszczenia konferencji na temat "Mycobacterial Stress Response"
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Bermudez, L., R. Rojony, A. Campeau, J. Wozniak, D. Gonzalez i L. Danelishvili. "Analysis of Mycobacterium Abscessus Subsp. Abscessus Proteome Upon Exposure to Stress Conditions Reveals Common Response Among Pathogenic Mycobacteria". W American Thoracic Society 2020 International Conference, May 15-20, 2020 - Philadelphia, PA. American Thoracic Society, 2020. http://dx.doi.org/10.1164/ajrccm-conference.2020.201.1_meetingabstracts.a6102.
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Bercovier, Herve, Raul Barletta i Shlomo Sela. Characterization and Immunogenicity of Mycobacterium paratuberculosis Secreted and Cellular Proteins. United States Department of Agriculture, styczeń 1996. http://dx.doi.org/10.32747/1996.7573078.bard.
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Our long-term goal is to develop an efficient acellular vaccine against paratuberculosis based on protein antigen(s). A prerequisite to achieve this goal is to analyze and characterize Mycobacterium paratuberculosis (Mpt) secreted and cellular proteins eliciting a protective immune response. In the context of this general objective, we proposed to identify, clone, produce, and characterize: the Mpt 85B antigen and other Mpt immunoreactive secreted proteins, the Mpt L7/L12 ribosomal protein and other immunoreactive cellular proteins, Mpt protein determinants involved in invasion of epithelial cells, and Mpt protein antigens specifically expressed in macrophages. Paratuberculosis is still a very serious problem in Israel and in the USA. In the USA, a recent survey evaluated that 21.6% of the dairy herd were infected with Mpt resulting in 200-250 million dollars in annual losses. Very little is known on the virulence factors and on protective antigens of Mpt. At present, the only means of controlling this disease are culling or vaccination. The current vaccines do not allow a clear differentiation between infected and vaccinated animals. Our long-term goal is to develop an efficient acellular paratuberculosis vaccine based on Mpt protein antigen(s) compatible with diagnostic tests. To achieve this goal it is necessary to analyze and characterize secreted and cellular proteins candidate for such a vaccine. Representative Mpt libraries (shuttle plasmid and phage) were constructed and used to study Mpt genes and gene products described below and will be made available to other research groups. In addition, two approaches were performed which did not yield the expected results. Mav or Mpt DNA genes that confer upon Msg or E. coli the ability to invade and/or survive within HEp-2 cells were not identified. Likewise, we were unable to characterize the 34-39 kDa induced secreted proteins induced by stress factors due to technical difficulties inherent to the complexity of the media needed to support substantial M. pt growth. We identified, isolated, sequenced five Mpt proteins and expressed four of them as recombinant proteins that allowed the study of their immunological properties in sensitized mice. The AphC protein, found to be up regulated by low iron environment, and the SOD protein are both involved in protecting mycobacteria against damage and killing by reactive oxygen (Sod) and nitrogen (AhpC) intermediates, the main bactericidal mechanisms of phagocytic cells. SOD and L7/L12 ribosomal proteins are structural proteins constitutively expressed. 85B and CFP20 are both secreted proteins. SOD, L7/L12, 85B and CFP20 were shown to induce a Th1 response in immunized mice whereas AphC was shown by others to have a similar activity. These proteins did not interfere with the DTH reaction of naturally infected cows. Cellular immunity provides protection in mycobacterial infections, therefore molecules inducing cellular immunity and preferentially a Th1 pathway will be the best candidate for the development of an acellular vaccine. The proteins characterized in this grant that induce a cell-mediated immunity and seem compatible with diagnostic tests, are good candidates for the construction of a future acellular vaccine.