Triphosphate Synthase Research Articles

Monitoring the dielectric response of single cells following mitochondrial adenosine triphosphate synthase inhibition by oligomycin using a dielectrophoretic cytometer.

One of the main uses of adenosine triphosphate (ATP) within mammalian cells is powering the Na(+)/K(+) ATPase pumps used to maintain ion concentrations within the cell. Since ion concentrations determine the cytoplasm conductivity, ATP concentration is expected to play a key role in controlling the cytoplasm conductivity. The two major ATP production pathways within cells are via glycolysis within the cytoplasm and via the electron transport chain within the mitochondria. In this work, a differential detector combined with dielectrophoretic (DEP) translation in a microfluidic channel was employed to observe single cell changes in the cytoplasm conductivity. The DEP response was made sensitive to changes in cytoplasm conductivity by measuring DEP response versus media conductivity and using double shell models to choose appropriate frequencies and media conductivity. Dielectric response of Chinese hamster ovary (CHO) cells was monitored following inhibition of the mitochondria ATP production by treatment with oligomycin. We show that in CHO cells following exposure to oligomycin (8 μg/ml) the cytoplasm conductivity drops, with the majority of the change occurring within 50 min. This work demonstrates that dielectric effects due to changes in ATP production can be observed at the single cell level.

Tauroursodeoxycholic acid increases neural stem cell pool and neuronal conversion by regulating mitochondria-cell cycle retrograde signaling

The low survival and differentiation rates of stem cells after either transplantation or neural injury have been a major concern of stem cell-based therapy. Thus, further understanding long-term survival and differentiation of stem cells may uncover new targets for discovery and development of novel therapeutic approaches. We have previously described the impact of mitochondrial apoptosis-related events in modulating neural stem cell (NSC) fate. In addition, the endogenous bile acid, tauroursodeoxycholic acid (TUDCA) was shown to be neuroprotective in several animal models of neurodegenerative disorders by acting as an anti-apoptotic and anti-oxidant molecule at the mitochondrial level. Here, we hypothesize that TUDCA might also play a role on NSC fate decision. We found that TUDCA prevents mitochondrial apoptotic events typical of early-stage mouse NSC differentiation, preserves mitochondrial integrity and function, while enhancing self-renewal potential and accelerating cell cycle exit of NSCs. Interestingly, TUDCA prevention of mitochondrial alterations interfered with NSC differentiation potential by favoring neuronal rather than astroglial conversion. Finally, inhibition of mitochondrial reactive oxygen species (mtROS) scavenger and adenosine triphosphate (ATP) synthase revealed that the effect of TUDCA is dependent on mtROS and ATP regulation levels. Collectively, these data underline the importance of mitochondrial stress control of NSC fate decision and support a new role for TUDCA in this process.

Reciprocal activation between ATPase inhibitory factor 1 and NF-κB drives hepatocellular carcinoma angiogenesis and metastasis.

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent extrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effects of ATPase inhibitory factor 1 (IF1), an inhibitor of the mitochondrial H(+)-adenosine triphosphate (ATP) synthase, on HCC angiogenesis and metastasis. We found that increased expression of IF1 in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and metastasis, expressed high levels of IF1. Invasive tumors overexpressing IF1 were featured by active epithelial-mesenchymal transition (EMT) and increased angiogenesis, whereas silencing IF1 expression attenuated EMT and invasion of HCC cells. Mechanistically, IF1 promoted Snai1 and vascular endothelial growth factor (VEGF) expression by way of activating nuclear factor kappa B (NF-κB) signaling, which depended on the binding of tumor necrosis factor (TNF) receptor-associated factor 1 (TRAF1) to NF-κB-inducing kinase (NIK) and the disruption of NIK association with the TRAF2-cIAP2 complex. Suppression of the NF-κB pathway interfered with IF1-mediated EMT and invasion. Chromatin immunoprecipitation assay showed that NF-κB can bind to the Snai1 promoter and trigger its transcription. IF1 was directly transcribed by NF-κB, thus forming a positive feedback signaling loop. There was a significant correlation between IF1 expression and pp65 levels in a cohort of HCC biopsies, and the combination of these two parameters was a more powerful predictor of poor prognosis. IF1 promotes HCC angiogenesis and metastasis by up-regulation of Snai1 and VEGF transcription, thereby providing new insight into HCC progression and IF1 function. (Hepatology 2014;60:1659-1673).

Primary Microcephaly, Muscle Hypotonia, and Global Developmental Delay: A Case of an Exceptional Clinical Manifestation of Maternally Inherited Mitochondrial T8993T>G Mutation in MT-ATP6 Gene

Objective: The m.8993T>G mutation in the gene MT-ATP6 (MIM:516060) which encodes subunit six of complex V (adenosine triphosphate synthase) of the mitochondrial respiratory chain is one of the more common disease associated mutations in mitochondrial DNA (mtDNA). The mutation is heteroplasmic, occurring together with wild-type mtDNA in affected individuals, and is associated with two different clinical phenotypes: NARP (neuropathy, ataxia, and retinitis pigmentosa) or MILS (maternally inherited Leigh syndrome), with the latter usually associated with a high mutation load.

Bedaquiline: a novel antitubercular agent for the treatment of multidrug-resistant tuberculosis.

Bedaquiline is a diarylquinoline antitubercular drug with a novel mechanism of action against Mycobacterium tuberculosis. Bedaquiline works by inhibiting bacterial adenosine triphosphate (ATP) synthase and represents the first novel class of antituberculosis agents in more than 40 years. Bedaquiline is indicated for the treatment of multidrug-resistant tuberculosis (MDR TB) in combination with at least three other antitubercular drugs when no other effective regimen is available. The recommended bedaquiline dosage is 400 mg orally once/day for 2 weeks followed by 200 mg orally 3 times/week for 22 weeks. Bedaquiline should be administered with food, which increases the bioavailability 2-fold. Bedaquiline is metabolized by cytochrome P450 isoenzyme 3A4 and is impacted by both inducers and inhibitors of this isoenzyme. Concentration-dependent bactericidal activity was observed in laboratory and murine studies. Accelerated approval was granted in the United States and European Union based on the results of two phase IIb clinical studies that used sputum culture clearance as a surrogate end point for clinical efficacy. These studies showed greater sputum culture clearance up to week 24 for the bedaquiline group compared with placebo. Common adverse events in clinical trials included nausea, arthralgia, and headache. Serious adverse events included elevated serum transaminase levels and rate-corrected QT-interval prolongation. Unexplained higher mortality was seen in patients receiving bedaquiline versus those receiving placebo. Bedaquiline is a novel agent with a unique mechanism of action and has the potential to meet a great need in patients with MDR TB who have no other treatment options. Due to safety concerns and limited clinical information, phase III trials are needed to fully determine its place in therapy.

Protein carbonylation associated to high-fat, high-sucrose diet and its metabolic effects

The present research draws a map of the characteristic carbonylation of proteins in rats fed high-caloric diets with the aim of providing a new insight of the pathogenesis of metabolic diseases derived from the high consumption of fat and refined carbohydrates. Protein carbonylation was analyzed in plasma, liver and skeletal muscle of Sprague–Dawley rats fed a high-fat, high-sucrose (HFHS) diet by a proteomics approach based on carbonyl-specific fluorescence-labeling, gel electrophoresis and mass spectrometry. Oxidized proteins along with specific sites of oxidative damage were identified and discussed to illustrate the consequences of protein oxidation. The results indicated that long-term HFHS consumption increased protein oxidation in plasma and liver; meanwhile, protein carbonyls from skeletal muscle did not change. The increment of carbonylation by HFHS diet was singularly selective on specific target proteins: albumin from plasma and liver, and hepatic proteins such as mitochondrial carbamoyl-phosphate synthase (ammonia), mitochondrial aldehyde dehydrogenase, argininosuccinate synthetase, regucalcin, mitochondrial adenosine triphosphate synthase subunit beta, actin cytoplasmic 1 and mitochondrial glutamate dehydrogenase 1. The possible consequences that these specific protein carbonylations have on the excessive weight gain, insulin resistance and nonalcoholic fatty liver disease resulting from HFHS diet consumption are discussed.

Electron cryomicroscopy of rotary ATPases

Ion-translocating rotary ATPases serve either as adenosine triphosphate (ATP) synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. Our group uses electron cryomicroscopy (cryo-EM) of single protein particles to study the structures of rotary ATPases. We also work to develop new methods for cryo-EM to facilitate these studies. The structures that we have obtained have illuminated several aspects of how structure relates to function in these remarkable molecular motors.

Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity.

Adenosine triphosphate (ATP) synthase β, the catalytic subunit of mitochondrial complex V, synthesizes ATP. We show that ATP synthase β is deacetylated by a human nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies displayed increased acetylation of specific Lys residues in ATP synthase β and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase β, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, we unravel a ceramide-NAD(+)-sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD(+) and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome.

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt-stress-tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3-O-methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.

Cardiolipin plays a role in KCN-induced necrosis

Cardiolipin (CL) is a unique anionic, dimeric phospholipid found almost exclusively in the inner mitochondrial membrane and is essential for the function of numerous enzymes that are involved in mitochondrial energy metabolism. While the role of cardiolipin in apoptosis is well established, its involvement in necrosis is enigmatic. In the present study, KCN-induced necrosis in U937 cells was used as an experimental model to assess the role of CL in necrosis. KCN addition to U937 cells induced reactive oxygen species (ROS) formation, while the antioxidants inhibited necrosis, indicating that ROS play a role in KCN-induced cell death. Further, CL oxidation was confirmed by the monomer green fluorescence of 10-N-nonyl acridine orange (NAO) and by TLC. Utilizing the red fluorescence of the dimeric NAO, redistribution of CL in mitochondrial membrane during necrosis was revealed. We also showed that the catalytic activity of purified adenosine triphosphate (ATP) synthase complex, known to be modulated by cardiolipin, decreased following KCN treatment. All these events occurred at an early phase of the necrotic process prior to rupture of the cell membrane. Furthermore, CL-deficient HeLa cells were found to be resistant to KCN-induced necrosis as compared with the wild type cells. We suggest that KCN, an effective reversible inhibitor of cytochrome oxidase and thereby of the respiratory chain leads to ROS increase, which in turn oxidizes CL (amongst other membrane phospholipids) and leads to mitochondrial membrane lipid reorganization and loss of CL symmetry. Finally, the resistance of CL-deficient cells to necrosis further supports the notion that CL, which undergoes oxidation during necrotic cell death, is an integral part of the milieu of events taking place in mitochondria leading to membrane disorganization and mitochondrial dysfunction.

New drugs to treat multidrug-resistant tuberculosis: the case for bedaquiline.

Mycobacterium tuberculosis develops spontaneous resistance mutants to virtually every drug in use. Courses of therapy select for these mutants and drug-resistant organisms emerge. The development of drug-resistant organisms has reached the point that drug resistance now threatens to undermine global success against tuberculosis (TB). New drugs are needed. The last new class of drugs specifically developed for treatment of TB was the rifamycins over 40 years ago. New funding sources and the development of product development partnerships have energized the TB drug development effort. There are now more TB drugs in development than at any time in the past. The first of these drugs to be developed and marketed was bedaquiline. Bedaquiline has an entirely novel mechanism of action and so should be active against otherwise highly resistant organisms. It acts on the transmembrane component of adenosine triphosphate synthase and acts by preventing electron transport. This raises the exciting possibility that bedaquiline may be active against less metabolically active organisms. Drug–drug interactions between rifamycins and the cytochrome P450-3A system will limit bedaquiline’s utility and create complexity in treatment regimens. In clinical trials, treatment with bedaquiline added to a background multidrug-resistant TB regimen was associated with earlier culture conversion and higher cure rates, but there were unexplained excess deaths in the bedaquiline arms of these trials. Food and Drug Administration approved bedaquiline for the treatment of multidrug-resistant TB when an effective treatment regimen cannot otherwise be provided. They required a black box warning about excess deaths and require that a phase III trial be completed. A planned Phase III trial is being reorganized. While bedaquiline is an exciting drug and marks a dramatic moment in the history of TB treatment, its ultimate place in the anti-TB drug armamentarium is unclear pending the Phase III trial and the development of other new drugs that are in the pipeline.

Sensitivity of housekeeping genes in the suprachiasmatic nucleus of the mouse brain to diet and the daily light–dark cycle

The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day–night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light–dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light–dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies.

Immunology: When lymphocytes run out of steam.

The finding that absence of the enzyme CTPS1 underlies a form of human immunodeficiency highlights the role of metabolism in immune responses and suggests avenues for treating diseases such as leukaemia. See Letter p.288 A study of several unrelated families susceptible to recurrent viral diseases but in whom known primary immunodeficiencies had been excluded has led to the identification of a novel human inherited immunodeficiency, and the recognition of a molecular pathway required to mount an efficient immune response against pathogens. Loss-of-function mutations in the gene for cytidine 5′ triphosphate synthase 1 (CTPS1) are associated with severe and selective impairment of T-cell proliferation after antigen stimulation, identifying the enzyme as a critical checkpoint in adaptive immunity. CTPS1 deficiency does not induce other phenotype abnormalities, so these findings suggest that CTPS1 could be a viable target for therapeutics to inhibit unwanted adaptive responses.

Phyllanthus urinaria induces mitochondrial dysfunction in human osteosarcoma 143B cells associated with modulation of mitochondrial fission/fusion proteins

Phyllanthus urinaria (P. urinaria), a widely used herbal medicine, has been reported to possess various biological characteristics including anti-inflammation, anti-virus, anti-bacteria, anti-hepatotoxicity and anti-cancer. This study provides molecular evidence associated with the dynamics and organization of mitochondria in osteosarcoma 143B cells resulted from P urinaria. Herein, P. urinaria-induced cytotoxicity and ROS associated with the inhibition of mitochondrial membrane potential were reversed by N-acetylcysteine (NAC). The endogenous antioxidant enzymes such as manganese superoxide dismutase (MnSOD) and glutathione peroxidase (GPX1) were activated by P. urinaria, but not correlated to catalase. P. urinaria decreased mitochondrial respiration activity as well as respiratory chain enzymes and HIF-1α in osteosarcoma 143B cells. Additionally, both adenosine triphosphate (ATP) synthase activation and ATP production were suppressed by P. urinaria. We further investigated changes of mitochondrial dynamic in osteosarcoma 143B cells. P. urinaria indeed fragmented the mitochondrial network of osteosarcoma 143B cells. We found a significant decrease in optic atrophy type 1 (Opa1) and mitofusin 1 (Mfn1) related to fusion proteins as well as increase mitochondrial fission 1 protein (Fis1) related to fission protein. It indicated that P. urinaria modulated the mitochondrial dynamics via fusion and fission machinery. Altogether, this study offers the evidence that mitochondrial dysfunction with dynamic change is essential components for the anti-cancer mechanism elicited by P. urinaria.

Study of the effect of varicocelectomy on sperm proteins expression in patients with varicocele and poor sperm quality by using two-dimensional gel electrophoresis.

To compare proteomic profiles of spermatozoa from patients with varicocele and poor sperm quality before and after varicocelectomy. This work was designed as a prospective and observational study. The study was based on 20 men with varicocele grade 3 and poor sperm quality undergoing varicocelectomy at the Fertility Unit of Royan institute in 2009. Two semen samples were collected, one before varicocelectomy and the other after surgery. Protein separation was done by two-dimensional protein electrophoresis, and analyzed by gel densitometry and mass spectrometry. Differential sperm protein expression levels were measured by gel densitometry. Comparison of the sperm parameters showed that sperm motility and concentration were increased after varicocelectomy. At the level of protein, a total of 3 protein spots were identified whose expression was significantly lower in sperm samples before varicocelectomy compared with after surgery including heat shock protein A5 (HSPA5), superoxide dismutase 1 (SOD1) and δ-subunit of the catalytic core of mitochondrial adenosine triphosphate synthase (ATP5D). High grade varicocoele affects sperm protein expression presumably because of increasing testicular temperature. These proteins play essential roles in sperm production, DNA integrity protection, and sperm motility. This novel study demonstrates that varicocelectomy can improve both sperm quality and proteins expression.

SIRT3 Deacetylates ATP Synthase F1 Complex Proteins in Response to Nutrient- and Exercise-Induced Stress

Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F1 proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP(K139) directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins.

Gene expression profiles of entorhinal cortex in Alzheimer's disease.

The incidence of Alzheimer's disease (AD) has been increasing in the recent years but the underlying mechanisms remain uncertain. This study aimed to analyze the differentially expressed genes (DEGs) in entorhinal cortex with AD and identify featured genes related to AD. Gene expression profile GSE5281 was downloaded from Gene Expression Omnibus, including 10 AD and 13 control samples. Differentially expressed genes were identified by Student t test including 119 upregulated and 591 downregulated DEGs. Then, we obtained 14 enrichment Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways among which 11 pathways were significantly enriched (adjusted P value < .05). The KEGG pathway network which was constructed by 14 KEGG pathways showed that 6-phosphofructokinase, muscle type, phosphoglucomutase 1, aldolase A, and adolase C had high degree. Glycometabolism pathways network which was constructed by 4 glycometabolism pathways showed that adenosine triphosphate (ATP) synthase, H+transporting, mitochondrial F1 complex ATP5B, ATP5C1, ATP5D, and ATP5G1 had high degree related to ATP metabolism. These findings suggested that these genes with high degree may be the underlying potential therapeutic targets for AD.

Regulation of the biogenesis of OXPHOS complexes in cell transition from replicating to quiescent state: Involvement of PKA and effect of hydroxytyrosol

A study is presented on the expression of mitochondrial oxidative phosphorylation complexes in exponentially growing and serum-starved, quiescent human fibroblast cultures. The functional levels of respiratory complexes I and III and complex V (adenosine triphosphate (ATP) synthase) were found to be severely depressed in serum-starved fibroblasts. The depression of oxidative phosphorylation system (OXPHOS) complexes was associated with reduced levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and the down-stream nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factors (TFAM). In serum-starved fibroblasts decrease of the catalytic activity of AMP cyclic dependent protein kinase (PKA) and phosphorylation of cAMP response element-binding protein (CREB), the transcription coactivator of the PGC-1α gene, was found. Hydroxytyrosol prevented the decline in the expression of the PGC-1α transcription cascade of OXPHOS complexes in serum-starved fibroblast cultures. The positive effect of HT was associated with activation of PKA and CREB phosphorylation. These results show involvement of PKA, CREB and PGC-1α in the regulation of OXPHOS in cell transition from the replicating to the quiescent state.

Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2

The cytidine-5'-triphosphate (CTP) synthase (EC 6.4.3.2) gene (pyrG) was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 (Ago). The gene is 1590 bp in length and encodes a protein of 530 amino acids, with a molecular mass of 59.5 kDa. The amino acid sequence of CTP synthase shares approximately 90%-94% similarity to Bacillus sp., and it belongs to the triad glutamine amidotransferases, which utilize a Cys-His-Glu triad for activity. Multiple sequence alignments revealed that the enzyme includes conserved amino acids responsible for catalytic activity and the binding of a divalent metal ion (Mg+2). AgoCTP synthase (AgoG2CTPs) was overproduced in Escherichia coli BL21 (DE3) pLysS as recombinant and purified by nickel affinity chromatography. Its biochemical characterization showed that the enzyme had maximal activity at pH 9.0-10.0 and 65 °C. Km, Vmax, and kcat were found to be approximately 12.415 mM, 0.381 U/L, and 0.762 s-1 at 65 °C, respectively. CTP synthase promotes the formation of CTP in dividing cells and is a recognized target for anticancer and antibacterial drugs. The results obtained from this study can be improved upon with the use of different species and substrates.

Coupling reporter expression to respiration detects active as well as dormant mycobacteria in vitro and in mouse tissues

BackgroundMycobacterium tuberculosis is known to slow down its transcriptional activity during dormancy. Hence, while using reporter strains, it is important to couple the reporter gene to a promoter that is strong and sensitive both in active and dormant M. tuberculosis. Since respiration is an indispensable process even in dormant bacteria, validation of the promoters of respiratory chain genes – type II NADH dehydrogenase (Pndh) and adenosine triphosphate (ATP) synthase operon (Patps) – of MTB was undertaken for this purpose. MethodsPutative promoter containing sequences were cloned upstream of a red fluorescent protein (RFP) gene. Mycobacterium smegmatis or M. tuberculosis carrying episomal constructs were validated for growth, fitness and fluorescence in different models in vitro and in vivo. ResultsEither promoter can drive stable and strong expression of RFP in actively growing and dormant M. smegmatis in vitro without significantly affecting growth or viability. Fluorescence due to Pndh and Patps was significantly higher than Phsp60. The fitness of M. tuberculosis H37Rv counterparts was unaffected inside J774 macrophages. In immunocompetent mice, despite an initial attenuation in the lungs, both strains reached loads similar to wild type during chronic infection. In the spleen, the fluorescent strain counts were similar to wild type counts throughout. RFP fluorescence in tissue homogenates was more homogenous among mice due to Pndh compared with Patps. ConclusionsCoupling an appropriate reporter to the promoter of ndh-2 gene of M. tuberculosis can make the reporter expression respiration sensitive and thereby reliably detect both active and dormant populations of the reporter strain.