scholarly journals mRNA Degradation Rates Are Coupled to Metabolic Status inMycobacterium smegmatis

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Diego A. Vargas-Blanco ◽  
Ying Zhou ◽  
L. Gregory Zamalloa ◽  
Tim Antonelli ◽  
Scarlet S. Shell
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mrna Stability ◽  
Mrna Degradation ◽  
Metabolic Status ◽  
Metabolic Adaptation ◽  
Content Type ◽  
Mrna Stabilization ◽  
Growth Status ◽  
Energy Stress ◽  
Multiple Drugs

ABSTRACTThe success ofMycobacterium tuberculosisas a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states,M. tuberculosiscan tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, includingM. tuberculosis, have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remain unknown. To better understand the biology of nongrowing mycobacteria, we sought to identify the mechanistic basis of mRNA stabilization in the nonpathogenic modelMycobacterium smegmatis. We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global stabilization was rapidly reversed when hypoxia-adapted cultures were reexposed to oxygen, even in the absence of new transcription. The stringent response and RNase levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activities of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status but rather is dependent on the status of energy metabolism.IMPORTANCEThe logistics of tuberculosis therapy are difficult, requiring multiple drugs for many months.Mycobacterium tuberculosissurvives in part by entering nongrowing states in which it is metabolically less active and thus less susceptible to antibiotics. Basic knowledge on howM. tuberculosissurvives during these low-metabolism states is incomplete, and we hypothesize that optimized energy resource management is important. Here, we report that slowed mRNA turnover is a common feature of mycobacteria under energy stress but is not dependent on the mechanisms that have generally been postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rateper se. Our findings suggest a need to reorient studies of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

scholarly journals mRNA Degradation Rates Are Coupled to Metabolic Status in Mycobacteria

2019 ◽  
Author(s):  
Diego A. Vargas-Blanco ◽  
Ying Zhou ◽  
Luis Gutierrez Zamalloa ◽  
Tim Antonelli ◽  
Scarlet S. Shell
Keyword(s):  
Mrna Stability ◽  
Mrna Degradation ◽  
Metabolic Status ◽  
Basic Knowledge ◽  
Metabolic Adaptation ◽  
Nutrient Deprivation ◽  
Mrna Stabilization ◽  
Growth Status ◽  
Metabolic States ◽  
Multiple Drugs

ABSTRACTThe success ofMycobacterium tuberculosis(Mtb) as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering non-growing states. In these low-metabolic states, Mtb can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including Mtb, have been shown to reduce their rates of mRNA degradation under growth limitation and stress. While the existence of this response appears to be conserved across species, the underlying bacterial mRNA stabilization mechanisms remains unknown. To better understand the biology of non-growing mycobacteria, we sought to identify the mechanisms by which mRNA stabilization occurs using the non-pathogenic modelMycobacterium smegmatis. We found that mRNA half-life was responsive to energy stress, with carbon starvation and hypoxia causing global mRNA stabilization. This global mRNA stabilization was rapidly reversed when hypoxia-adapted cultures were re-exposed to oxygen, even in the absence of new transcription. The stringent response and RNase protein levels did not explain mRNA stabilization, nor did transcript abundance. This led us to hypothesize that metabolic changes during growth cessation impact the activity of degradation proteins, increasing mRNA stability. Indeed, bedaquiline and isoniazid, two drugs with opposing effects on cellular energy status, had opposite effects on mRNA half-lives in growth-arrested cells. Taken together, our results indicate that mRNA stability in mycobacteria is not directly regulated by growth status, but rather seems to be dependent on the status of energy metabolism.IMPORTANCEThe logistics of treating tuberculosis are difficult, requiring multiple drugs for at least six months. Mtb is able to survive within the human host in part by entering non-growing states in which it is metabolically less active, thus rendering it less susceptible to antibiotics. Basic knowledge on how Mtb survives during these low-metabolic states is incomplete, and we postulate that optimized energy resource management –such as transcriptome stabilization—is important for survival. Here we report that mRNA stabilization (increased mRNA half-lives) is a common feature of mycobacteria under stress (e.g. hypoxia and nutrient deprivation) but is not dependent on the mechanisms that have been most often postulated in the literature. Finally, we found that mRNA stability and growth status can be decoupled by a drug that causes growth arrest but increases metabolic activity, indicating that mRNA stability responds to metabolic status rather than to growth rate changes per se. Our findings suggest a need to re-orient the study of global mRNA stabilization to identify novel mechanisms that are presumably responsible.

scholarly journals Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus

2015 ◽  
Vol 59 (10) ◽  
pp. 6561-6567 ◽  
Author(s):  
Amit Kaushik ◽  
Nayani Makkar ◽  
Pooja Pandey ◽  
Nicole Parrish ◽  
Urvashi Singh ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Utility ◽  
Mycobacterium Abscessus ◽  
Synergistic Activity ◽  
Drug Resistant ◽  
Content Type ◽  
Tb Treatment ◽  
Resistant Mutants ◽  
Multiple Drugs ◽  
Spontaneous Mutants

ABSTRACTAn effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities inMycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitiveMycobacterium tuberculosisbut also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity againstMycobacterium abscessus. Although the MICs of these three drugs alone againstM. abscessusare too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treatMycobacterium abscessusinfection, especially in cystic fibrosis patients.

scholarly journals From infection niche to therapeutic target: the intracellular lifestyle of Mycobacterium tuberculosis

Microbiology ◽  
2021 ◽  
Vol 167 (4) ◽  
Author(s):  
Leah Isobella Rankine-Wilson ◽  
Tirosh Shapira ◽  
Carine Sao Emani ◽  
Yossef Av-Gay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Side Effects ◽  
Type Species ◽  
Life Stage ◽  
Maturation Process ◽  
Human Pathogen ◽  
Phagosome Maturation ◽  
Content Type ◽  
The Past ◽  
Multiple Drugs

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen killing millions of people annually. Treatment for tuberculosis is lengthy and complicated, involving multiple drugs and often resulting in serious side effects and non-compliance. Mtb has developed numerous complex mechanisms enabling it to not only survive but replicate inside professional phagocytes. These mechanisms include, among others, overcoming the phagosome maturation process, inhibiting the acidification of the phagosome and inhibiting apoptosis. Within the past decade, technologies have been developed that enable a more accurate understanding of Mtb physiology within its intracellular niche, paving the way for more clinically relevant drug-development programmes. Here we review the molecular biology of Mtb pathogenesis offering a unique perspective on the use and development of therapies that target Mtb during its intracellular life stage.

scholarly journals P38 activation induces the dissociation of tristetraprolin from Argonaute 2 to increase ARE-mRNA stabilization

2018 ◽  
Vol 29 (8) ◽  
pp. 988-1002 ◽  
Author(s):  
Mei-Yan Qi ◽  
Jing-Wen Song ◽  
Zhuo Zhang ◽  
Shuang Huang ◽  
Qing Jing
Keyword(s):  
Mrna Stability ◽  
Mrna Decay ◽  
Mrna Degradation ◽  
Processing Bodies ◽  
Mrna Stabilization ◽  
Argonaute 2 ◽  
Core Member ◽  
Mrna Granules

Tristetraprolin (TTP) destabilizes AU-rich element (ARE)-containing mRNA by directly binding with their 3′UTR. P38 stimulation substantially increases ARE-mRNA stability, at least through repressing TTP. However, the mechanism by which P38 keeps TTP inactive has not been fully understood. TTP and ARE-mRNA localize to processing bodies (PBs), the mRNA granules associated with mRNA silencing. Here, we detected the influence of P38 on TTP localization within PBs and found that P38 regulates TTP localization within PBs. Through luciferase-based systems, we demonstrated that PBs depletion significantly increased ARE-mRNA stability inhibited by TTP. Additionally, we provided evidence that the microRNA-induced silencing complex (miRISC) core member Ago2 is required for TTP distribution within PBs. Importantly, the cooperation of TTP and Ago2 is a prerequisite for effective ARE-mRNA degradation. Moreover, Dcp1a and Dcp2 act downstream of Ago2 and TTP engaging in ARE-mRNA decay. Finally, we demonstrated that P38 activation represses the interaction between TTP and Ago2 due to TTP phosphorylation, which impairs TTP localization within PBs and ARE-mRNA degradation. Collectively, our study revealed a novel mechanism through which P38 activation repressed the cooperation of TTP with Ago2, thus ensuring that ARE-mRNA does not associate with PBs and remains stable.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

scholarly journals A Novel 6-Benzyl Ether Benzoxaborole Is Active against Mycobacterium tuberculosisIn Vitro

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Nipul Patel ◽  
Theresa O'Malley ◽  
Yong-Kang Zhang ◽  
Yi Xia ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Medium ◽  
Inhibitory Concentration ◽  
Solid Medium ◽  
Eukaryotic Cells ◽  
Intracellular Bacteria ◽  
Benzyl Ether ◽  
Content Type ◽  
Resistant Mutants

ABSTRACT We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis. The compound had an MIC of 2 μM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 μM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 μM) without cytotoxicity against eukaryotic cells (IC50 > 100 μM). We isolated resistant mutants (MIC ≥ 100 μM), which had mutations in Rv1683, Rv3068c, and Rv0047c.

scholarly journals Defects in Mitochondrial and Peroxisomal β-Oxidation Influence Virulence in the Maize Pathogen Ustilago maydis

2012 ◽  
Vol 11 (8) ◽  
pp. 1055-1066 ◽  
Author(s):  
Matthias Kretschmer ◽  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Ustilago Maydis ◽  
Short Chain Fatty Acids ◽  
Phytopathogenic Fungi ◽  
Metabolic Adaptation ◽  
In Planta ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Content Type ◽  
C Terminus ◽  
Fungal Phytopathogens

ABSTRACTAn understanding of metabolic adaptation during the colonization of plants by phytopathogenic fungi is critical for developing strategies to protect crops. Lipids are abundant in plant tissues, and fungal phytopathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. Previously, we demonstrated a role for the peroxisomal β-oxidation enzyme Mfe2 in the filamentous growth, virulence, and sporulation of the maize pathogenUstilago maydis. However,mfe2mutants still caused disease symptoms, thus prompting a more detailed investigation of β-oxidation. We now demonstrate that a defect in thehad1gene encoding hydroxyacyl coenzyme A dehydrogenase for mitochondrial β-oxidation also influences virulence, although its paralog,had2, makes only a minor contribution. Additionally, we identified a gene encoding a polypeptide with similarity to the C terminus of Mfe2 and designated it Mfe2b; this gene makes a contribution to virulence only in the background of anmfe2Δ mutant. We also show that short-chain fatty acids induce cell death inU. maydisand that a block in β-oxidation leads to toxicity, likely because of the accumulation of toxic intermediates. Overall, this study reveals that β-oxidation has a complex influence on the formation of disease symptoms byU. maydisthat includes potential metabolic contributions to proliferationin plantaand an effect on virulence-related morphogenesis.

scholarly journals Characterization of T Cells Specific for CFP-10 and ESAT-6 in Mycobacterium tuberculosis-Infected Mauritian Cynomolgus Macaques

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Amy Ellis ◽  
Alexis Balgeman ◽  
Mark Rodgers ◽  
Cassaundra Updike ◽  
Jaime Tomko ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
Mhc Class Ii ◽  
Nonhuman Primates ◽  
Content Type ◽  
Host Immune Responses ◽  
Cell Responses ◽  
Cynomolgus Macaques ◽  
Mhc Haplotype

ABSTRACT Nonhuman primates can be used to study host immune responses to Mycobacterium tuberculosis. Mauritian cynomolgus macaques (MCMs) are a unique group of animals that have limited major histocompatibility complex (MHC) genetic diversity, such that MHC-identical animals can be infected with M. tuberculosis. Two MCMs homozygous for the relatively common M1 MHC haplotype were bronchoscopically infected with 41 CFU of the M. tuberculosis Erdman strain. Four other MCMs, which had at least one copy of the M1 MHC haplotype, were infected with a lower dose of 3 CFU M. tuberculosis. All animals mounted similar T-cell responses to CFP-10 and ESAT-6. Two epitopes in CFP-10 were characterized, and the MHC class II alleles restricting them were determined. A third epitope in CFP-10 was identified but exhibited promiscuous restriction. The CFP-10 and ESAT-6 antigenic regions targeted by T cells in MCMs were comparable to those seen in cases of human M. tuberculosis infection. Our data lay the foundation for generating tetrameric molecules to study epitope-specific CD4 T cells in M. tuberculosis-infected MCMs, which may guide future testing of tuberculosis vaccines in nonhuman primates.

scholarly journals Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Zhaojing Zong ◽  
Wei Jing ◽  
Jin Shi ◽  
Shu'an Wen ◽  
Tingting Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Novel Mutation ◽  
Multidrug Resistant ◽  
23S Rrna ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Significant Difference ◽  
Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

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