scholarly journals Genetic and metabolic regulation ofMycobacterium tuberculosisacid growth arrest

2017 ◽  
Author(s):  
Jacob J. Baker ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Metabolic Regulation ◽  
Isocitrate Lyase ◽  
Growth Arrest ◽  
Drug Tolerance ◽  
Acidic Ph ◽  
Acid Growth ◽  
Physiological Basis ◽  
Acidic Environments ◽  
Ph Dependent

AbstractMycobacterium tuberculosis(Mtb) senses and adapts to acidic environments during the course of infection. Acidic pH-dependent adaptations include the induction of metabolic genes associated with anaplerosis and growth arrest on specific carbon sources. In this study, reverse and forward genetic studies were undertaken to define new mechanisms underlying pH-dependent adaptations. Here we report that deletion of isocitrate lyase (icl1/2) or phosphoenolpyruvate carboxykinase (pckA) results in reduced growth at acidic pH and altered metabolite profiles, supporting that remodeling of anaplerotic metabolism is required for pH-dependent adaptation. Mtb cultured at pH 5.7 in minimal medium containing glycerol as a single carbon source exhibits an acid growth arrest phenotype, where the bacterium is non-replicating but viable and metabolically active. The bacterium uptakes and metabolizes glycerol and maintains ATP pools during acid growth arrest and becomes tolerant to detergent stress and the antibiotics isoniazid and rifampin. A forward genetic screen identified mutants that do not arrest their growth at acidic pH, including four enhanced acid growth (eag) mutants with three distinct mutations in the PPE gene MT3221. Overexpression of the MT3221(S211R) variant protein in wild type Mtb results in enhanced acid growth and reduced drug tolerance. Together, these findings provide new evidence for a genetic and physiological basis for acid growth arrest and support that growth arrest is an adaptive process and not simply a physiological limitation associated with acidic pH.Author SummaryThe bacteriumMycobacterium tuberculosis(Mtb) causes the disease tuberculosis in humans. During infection Mtb colonizes a variety of environments that have acidic environments and Mtb must adapt to these environments to cause disease. One of these adaptations is that Mtb slows and arrests its growth at acidic pH, and the goal of this study was to examine the genetics and physiology of these pH-dependent adaptations. We found that Mtb modifies its metabolism at acidic pH and that these adaptations are required for optimal growth. We also found that acidic pH and specific nutrient sources can promote the bacterium to enter a state of dormancy, called acid growth arrest, where the bacterium becomes tolerant to antibiotics. Mutants were identified that do not arrest their growth at acidic, revealing that acid growth arrest is a genetically controlled process. Overall, understanding how Mtb adapts to acidic pH has revealed pathway that are required for virulence and drug tolerance and thus may identify new targets for drug development that may function to shorten the course of TB therapy.

scholarly journals Remodeling of Mycobacterium tuberculosis lipids regulates prpCD during acid growth arrest

2019 ◽  
Author(s):  
Jacob J. Baker ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Carbon Source ◽  
Isocitrate Lyase ◽  
Cell Envelope ◽  
Transcriptional Profiling ◽  
Carbon Sources ◽  
Growth Arrest ◽  
Acidic Ph ◽  
Acid Growth ◽  
Metabolic Adaptations

AbstractMycobacterium tuberculosis (Mtb) establishes a state of non-replicating persistence when it is cultured at acidic pH with glycerol as a sole carbon source. Growth can be restored by spontaneous mutations in the ppe51 gene or supplementation with pyruvate, supporting that acid growth arrests is a genetically controlled, adaptive process and not simply a physiological limitation associated with acidic pH. Transcriptional profiling identified the methylcitrate synthase and methylcitrate dehydratase genes (prpC and prpD, respectively) as being selectively induced during acid growth arrest. prpCD along with isocitrate lyase (icl) enable Mtb to detoxify propionyl-CoA through the methylcitrate cycle. The goal of this study was to examine mechanisms underlying the regulation of prpCD during acid growth arrest. Induction of prpCD during acid growth arrest was reduced when the medium was supplemented with vitamin B12 (which enables an alternative propionate detoxification pathway) and enhanced in an icl mutant (which is required for the propionate detoxification), suggesting that Mtb is responding to elevated levels of propionyl-CoA during acidic growth arrest. We hypothesized that an endogenous source of propionyl-CoA generated during metabolism of methyl-branched lipids may be regulating prpCD. Using Mtb radiolabeled with 14C-propionate or 14C-acetate, it was observed that lipids are remodeled during acid growth arrest, with triacylglycerol being catabolized and sulfolipid and trehalose dimycolate being synthesized. Blocking TAG lipolysis using the lipase inhibitor tetrahydrolipstatin, resulted in enhanced prpC induction during acid growth arrest, suggesting that lipid remodeling may function, in part, to detoxify propionate. Notably, prpC was not induced during acid growth arrest when using lactate instead of glycerol. We propose that metabolism of glycerol at acidic pH may result in the accumulation of propionyl-CoA and that lipid remodeling may function as a detoxification mechanism.ImportanceDuring infection, Mycobacterium tuberculosis (Mtb) colonizes acidic environments, such as the macrophage phagosome and granuloma. Understanding regulatory and metabolic adaptations that occur in response to acidic pH can provide insights int0 mechanisms used by the bacterium to adapt to the host. We have previously shown that Mtb exhibits pH-dependent metabolic adaptations and requires anaplerotic enzymes, such as Icl1/2 and PckA, to grow optimally at acidic pH. Additionally, we have observed that Mtb can only grow on specific carbon sources at acidic pH. Together these findings show that Mtb integrates environmental pH and carbon source to regulate its metabolism. In this study, it is shown that Mtb remodels its lipids and modulates the expression of propionyl-CoA detoxifying genes prpCD when grown on glycerol at acidic pH. This finding suggests that lipid remodeling at acidic pH may contribute to detoxification of propionyl-CoA, by incorporating the metabolite into methyl-branched cell envelope lipids.

scholarly journals ppe51 variants promote non-replicating Mycobacterium tuberculosis to grow at acidic pH by selectively promoting glycerol uptake

2021 ◽  
Author(s):  
Shelby J. Dechow ◽  
Jacob J. Baker ◽  
Megan R. Murto ◽  
Robert B. Abramovitch
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Carbon Source ◽  
Specific Growth ◽  
Carbon Sources ◽  
Growth Arrest ◽  
Acidic Ph ◽  
Genetic Screens ◽  
Acid Growth ◽  
Forward Genetic Screens ◽  
Glycerol Uptake

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis exhibits carbon source specific growth restriction. When supplied glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acidic growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we conducted forward genetic screens that identified several Mtb mutants that could grow under these restrictive conditions. All of the mutants were mapped to the ppe51 gene and resulted in three amino acid substitution, S211R, E215K, and A228D. Expression of the PPE51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, enhanced acid growth (eag) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting ppe51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced in virulence in activated macrophages as compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the WT or S211R variants in the presence of glycerol, supporting the eag substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis.

scholarly journals Growth of Mycobacterium tuberculosis at acidic pH depends on lipid assimilation and is accompanied by reduced GAPDH activity

2021 ◽  
Vol 118 (32) ◽  
pp. e2024571118
Author(s):  
Alexandre Gouzy ◽  
Claire Healy ◽  
Katherine A. Black ◽  
Kyu Y. Rhee ◽  
Sabine Ehrt
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Oleic Acid ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Isocitrate Lyase ◽  
Acid Stress ◽  
Glycolytic Flux ◽  
Acidic Ph ◽  
Gapdh Activity ◽  
Cell Host Microbe

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199–3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250–258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

scholarly journals Acidic pH-dependent depletion ofMycobacterium tuberculosisthiol pools potentiates antibiotics and oxidizing agents

2016 ◽  
Author(s):  
Garry B. Coulson ◽  
Benjamin K. Johnson ◽  
Christopher J. Colvin ◽  
Robert J. Fillinger ◽  
Huiqing Zheng ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mycobacterium Tuberculosis ◽  
Bactericidal Activity ◽  
Metabolic Stress ◽  
Acidic Ph ◽  
Oxygen Species ◽  
Bacterial Killing ◽  
Oxidizing Agents ◽  
Intracellular Ros ◽  
Ph Dependent

SummaryMycobacterium tuberculosis(Mtb) must sense and adapt to immune pressures such as acidic pH and reactive oxygen species (ROS) during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here we report that the acidic pH-dependent compound AC2P36 depletes intracellular thiol pools, sensitizes Mtb to killing by acidic pH, and potentiates the bactericidal activity of isoniazid, clofazimine, and oxidizing agents. We show that the pHdependent activity of AC2P36 is associated with metabolic stress at acidic pH and a pHdependent accumulation of intracellular ROS. Mechanism of action studies show that AC2P36 directly depletes Mtb thiol pools. These data support a model where chemical depletion of Mtb thiol pools at acidic pH enhances sensitivity to oxidative damage, resulting in bacterial killing and potentiation of antibiotics.

scholarly journals Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Poushali Chakraborty ◽  
Sapna Bajeli ◽  
Deepak Kaushal ◽  
Bishan Dass Radotra ◽  
Ashwani Kumar
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune System ◽  
Biofilm Formation ◽  
Antimycobacterial Activity ◽  
Drug Tolerance ◽  
Host Immune System ◽  
Tissue Sections ◽  
Slow Growing

AbstractTuberculosis is a chronic disease that displays several features commonly associated with biofilm-associated infections: immune system evasion, antibiotic treatment failures, and recurrence of infection. However, although Mycobacterium tuberculosis (Mtb) can form cellulose-containing biofilms in vitro, it remains unclear whether biofilms are formed during infection in vivo. Here, we demonstrate the formation of Mtb biofilms in animal models of infection and in patients, and that biofilm formation can contribute to drug tolerance. First, we show that cellulose is also a structural component of the extracellular matrix of in vitro biofilms of fast and slow-growing nontuberculous mycobacteria. Then, we use cellulose as a biomarker to detect Mtb biofilms in the lungs of experimentally infected mice and non-human primates, as well as in lung tissue sections obtained from patients with tuberculosis. Mtb strains defective in biofilm formation are attenuated for survival in mice, suggesting that biofilms protect bacilli from the host immune system. Furthermore, the administration of nebulized cellulase enhances the antimycobacterial activity of isoniazid and rifampicin in infected mice, supporting a role for biofilms in phenotypic drug tolerance. Our findings thus indicate that Mtb biofilms are relevant to human tuberculosis.

scholarly journals Development of optimized formulation of liposome using 3-factor Box-Behnken Design

2021 ◽  
Vol 11 (3) ◽  
Author(s):  
Mahendra Chouhan ◽  
Rajesh Sharma ◽  
Kamlesh Dashora
Keyword(s):  
Response Surface ◽  
Polymer Coating ◽  
Drug Loading ◽  
Membrane System ◽  
Acidic Ph ◽  
Box Behnken Design ◽  
Vesicle Size ◽  
Surface Graph ◽  
Ph Dependent ◽  
Ph Sensitive Polymer

The objective of our present study is to optimize Chitosan coated liposomes formulation by Response surface methodology using 3-factor Box-Behnken Design. Different polymers based liposomes used for delivery of stable pH dependent formulation. Chitosan has been used as a pH sensitive polymer coating to target nanoparticles specifically to tumors which have a slightly acidic pH. Closed membrane system can accommodate amphiphilic or lipophilic drugs in vesicles. The optimized batch was formulated as a liposome delivery system and evaluation was done. To evaluate the unentrapped drug Shimadzu UV-Spectrometry at 228 nm was used and absorbance was noted. Response surface graph was prepared to predict value and the optimized formulation (Chitosan coated liposomes) can be used for loading of bio-active.The values of percent drug entrapment and average vesicle size were presented and found formulation F3&F5 were optimized for further evaluating on basis of particle size and drug loading.

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