Faculty Opinions recommendation of Carbon flux rerouting during Mycobacterium tuberculosis growth arrest.

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.

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Tween 80 induces a carbon flux rerouting in Mycobacterium tuberculosis

Journal of Microbiological Methods ◽

10.1016/j.mimet.2019.105795 ◽

2020 ◽

Vol 170 ◽

pp. 105795

Author(s):

Ray-Dean Pietersen ◽

Ilse du Preez ◽

Du Toit Loots ◽

Mari van Reenen ◽

Derylize Beukes ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Carbon Flux ◽

Tween 80

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The diacylglycerol acyltransferase Rv3371 of Mycobacterium tuberculosis is required for growth arrest and involved in stress-induced cell wall alterations

Tuberculosis ◽

10.1016/j.tube.2017.02.001 ◽

2017 ◽

Vol 104 ◽

pp. 8-19 ◽

Cited By ~ 5

Author(s):

Shivangi Rastogi ◽

Amit Kumar Singh ◽

Gyan Chandra ◽

Pragati Kushwaha ◽

Garima Pant ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Growth Arrest ◽

Diacylglycerol Acyltransferase

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Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages

Journal of Bacteriology ◽

10.1128/jb.01318-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1618-1630 ◽

Cited By ~ 72

Author(s):

Shaleen B. Korch ◽

Heidi Contreras ◽

Josephine E. Clark-Curtiss

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Protein Pair ◽

Growth Arrest ◽

Broth Culture ◽

Human Macrophages ◽

In Vivo Analysis ◽

Mycobacterial Growth

ABSTRACT Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages.

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Genetic and metabolic regulation ofMycobacterium tuberculosisacid growth arrest

10.1101/186551 ◽

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.

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Translation of a leaderless reporter is robust during exponential growth and well sustained during stress conditions in Mycobacterium tuberculosis

10.21203/rs.3.rs-365129/v1 ◽

2021 ◽

Author(s):

A. D. Grabowska ◽

N. Andreu ◽

T. Cortes

Keyword(s):

Mycobacterium Tuberculosis ◽

Translational Regulation ◽

Molecular Mechanisms ◽

Growth Arrest ◽

Optimal Growth ◽

Growth Conditions ◽

Stress Conditions ◽

Shine Dalgarno Sequence ◽

Overall Ratio

Abstract Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5’ leader region, which usually contains the Shine-Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins with secondary adaptive functions are predominantly leaderless and the overall ratio of leaderless to Shine-Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during stress conditions that induce growth arrest and during optimal growth conditions, is unclear. Here, using bioluminescent reporter strains, we detect robust leaderless translation during exponential in vitro growth and we show that leaderless translation is more stable than Shine-Dalgarno translation during adaptation to stress conditions. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine-Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are more stable than those of Shine-Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

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

10.1101/2021.05.19.444820 ◽

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.

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