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Mycobacterium tuberculosis can enter a dormant state enabling it to establish latent infections and to become tolerant to antibacterial drugs. Dormant bacteria’s physiology and the mechanism(s) used by bacteria to enter dormancy during infection are still unknown due to the lack of reliable animal models.

Summary Bacterial nutrition is an essential aspect of host–pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a ‘multi‐omics’ approach consisting of transposon‐directed insertion site sequencing (TraDIS), RNA‐seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry‐based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl‐CoA, the essential precursor for the biosynthesis of the odd‐chain fatty acids., Mycobacterium tuberculosis assimilates lactate and pyruvate utilising glyoxylate shunt and methylcitrate cycle, two pathways traditionally associated with fatty acid metabolism in bacteria. Surprisingly, we found that M. tuberculosis synthesises propionyl‐CoA by a reverse methylcitrate cycle and another partially characterised (in this study) pathway. Our results point to a re‐interpretation of the mechanistic role and utilisation of these pathways by Mtb during infection, i.e., their potential reversibility and importance during lactate and pyruvate metabolism in vivo.

Bacterial nutrition is a fundamental aspect of pathogenesis. While the host environment is in principle nutrient-rich, hosts have evolved strategies to interfere with nutrient acquisition by pathogens. In turn, pathogens have developed mechanisms to circumvent these restrictions. Changing the availability of bioavailable metal ions is a common strategy used by hosts to limit bacterial replication. Macrophages and neutrophils withhold iron, manganese, and zinc ions to starve bacteria. Alternatively, they can release manganese, zinc, and copper ions to intoxicate microorganisms. Metals are essential micronutrients and participate in catalysis, macromolecular structure, and signalling. This review summarises our current understanding of how central carbon metabolism in pathogens adapts to local fluctuations in free metal ion concentrations. We focus on the transcriptomics and proteomics data produced in studies of the iron-sparing response in Mycobacterium tuberculosis , the etiological agent of tuberculosis, and consequently generate a hypothetical model linking trehalose accumulation, succinate secretion and substrate-level phosphorylation in iron-starved M. tuberculosis . This review also aims to highlight a large gap in our knowledge of pathogen physiology: the interplay between metal homeostasis and central carbon metabolism, two cellular processes which are usually studied separately. Integrating metabolism and metal biology would allow the discovery of new weaknesses in bacterial physiology, leading to the development of novel and improved antibacterial therapies.

Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential gamma-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported gamma-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of beta-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that gamma-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that gamma-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active gamma-aminobutyric acid transport protein from mycobacteria. IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport beta-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.

The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species., Characterizing the mycobacterial transporters involved in the uptake and/or catabolism of host-derived nutrients required by mycobacteria may identify novel drug targets against tuberculosis. Here, we identify and characterize a member of the amino acid-polyamine-organocation superfamily, a potential γ-aminobutyric acid (GABA) transport protein, GabP, from Mycobacterium smegmatis. The protein was expressed to a level allowing its purification to homogeneity, and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS) analysis of the purified protein showed that it was dimeric. We showed that GabP transported γ-aminobutyric acid both in vitro and when overexpressed in E. coli. Additionally, transport was greatly reduced in the presence of β-alanine, suggesting it could be either a substrate or inhibitor of GabP. Using GabP reconstituted into proteoliposomes, we demonstrated that γ-aminobutyric acid uptake is driven by the sodium gradient and is stimulated by membrane potential. Molecular docking showed that γ-aminobutyric acid binds MsGabP, another Mycobacterium smegmatis putative GabP, and the Mycobacterium tuberculosis homologue in the same manner. This study represents the first expression, purification, and characterization of an active γ-aminobutyric acid transport protein from mycobacteria. IMPORTANCE The spread of multidrug-resistant tuberculosis increases its global health impact in humans. As there is transmission both to and from animals, the spread of the disease also increases its effects in a broad range of animal species. Identifying new mycobacterial transporters will enhance our understanding of mycobacterial physiology and, furthermore, provides new drug targets. Our target protein is the gene product of msmeg_6196, annotated as GABA permease, from Mycobacterium smegmatis strain MC2 155. Our current study demonstrates it is a sodium-dependent GABA transporter that may also transport β-alanine. As GABA may well be an essential nutrient for mycobacterial metabolism inside the host, this could be an attractive target for the development of new drugs against tuberculosis.

Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo. One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants.

Bacterial metabolism is fundamental to survival and pathogenesis. We explore how Mycobacterium tuberculosis utilises amino acids as nitrogen sources, using a combination of bacterial physiology and stable isotope tracing coupled to mass spectrometry metabolomics methods. Our results define core properties of the nitrogen metabolic network from M. tuberculosis, such as: (i) the lack of homeostatic control of certain amino acid pool sizes; (ii) similar rates of utilisation of different amino acids as sole nitrogen sources; (iii) improved nitrogen utilisation from amino acids compared to ammonium; and (iv) co-metabolism of nitrogen sources. Finally, we discover that alanine dehydrogenase is involved in ammonium assimilation in M. tuberculosis, in addition to its essential role in alanine utilisation as a nitrogen source. This study represents the first in-depth analysis of nitrogen source utilisation by M. tuberculosis and reveals a flexible metabolic network with characteristics that are likely a product of evolution in the human host., eLife digest Tuberculosis is an infectious disease caused by a bacterium called Mycobacterium tuberculosis. It is currently the leading cause of death by a single microbe worldwide, claiming the lives of 1.5 million people annually. The disease is difficult to cure, as many strains of the bacterium have developed resistance to the main drugs used to treat the infection. This leaves physicians with few options to treat tuberculosis and control its spread. The spread of these drug-resistant strains is a major global public health problem. New strategies that do not lead to drug resistance are needed. One possibility would be to starve the bacterium. Like all living things, M. tuberculosis must eat to survive and spread. Right now, scientists do not know much about how this microbe eats. However, they do know that it needs nitrogen – an essential part of DNA, RNA, and proteins – to survive. Most bacteria like to consume ammonium as their main nitrogen source, but they may also use select amino acids as a nitrogen source. Now, Agapova et al. show that M. tuberculosis is not a picky eater. In the experiments, the bacteria were fed different nitrogen sources. Then, they tracked how well the bacteria grew. The experiments showed that M. tuberculosis happily eats many different amino acids and may use more than one as a nitrogen source at a time. It does not tightly control its stockpile of nitrogen sources the way other bacteria do, or use ammonium very efficiently. This suggests that M. tuberculosis has evolved to be very flexible in its dietary habits, which may explain why these bacteria can thrive in the varied environments within the human body. Knowing exactly how M. tuberculosis acquires and uses nitrogen may help scientists design ways to thwart the process and starve the bacteria.

In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.

Genetic manipulation of mycobacteria still represents a serious challenge due to the lack of tools and selection markers. In this report, we describe the development of an intrinsically unstable excisable cassette for introduction of unmarked mutations in both Mycobacterium smegmatis and Mycobacterium tuberculosis .

Recently, a novel type of secretory pathway, type VII secretion systems (T7SSs), has been characterized in mycobacteria. The chromosomes of Mycobacterium tuberculosis and Mycobacterium bovis encode five T7SSs (ESX-1 to ESX-5). The best characterized of them, ESX-1, is involved in host-pathogen interactions, and its deletion is one of the main causes of M. bovis BCG attenuation. Another T7SS, ESX-3, has been previously shown to be transcriptionally controlled by the zinc uptake repressor (Zur) and by the iron-dependent transcriptional repressor (IdeR), suggesting that it might be involved in zinc and iron homeostasis. In this study, we characterized an M. tuberculosis conditional mutant in which transcription of the ESX-3 gene cluster can be downregulated by anhydrotetracycline. We showed that this T7SS is essential for growth and that this phenotype can be complemented by zinc, iron, or supernatant from a wild-type parental strain culture, demonstrating that the ESX-3 secretion system is responsible for the secretion of some soluble factor(s) required for growth that is probably involved in optimal iron and zinc uptake.

Purpose: Long-term space exploration requires biological life support systems capable of coping with the deleterious space environment. The use of oxygenic photosynthetic microorganisms represents an intriguing topic in this context, mainly from the point of view of food and O2 production. The aim of the present study was to assess the effects of space ionizing radiation exposure on the photosynthetic activity of various microorganisms. Materials and methods: Ground-based irradiation experiments were performed using fast neutrons and gamma rays on microorganisms maintained at various light conditions. A stratospheric balloon and a European Space Agency (ESA) flight facility were used to deliver organisms to space at the altitude of 38 and 300 km, respectively. During the balloon flight, the fluorescence activity of the organisms was real-time monitored by means of a special biosensor. Results: The quantum yield of Photosystem II (PSII), measured directly in flight, varied among the microorganisms depending on the light conditions. Darkness and irradiation of cells at 120 and 180 ?mol m-2 s-1 enhanced the radiation-induced inhibition of photosynthetic activity, while exposure to weaker light irradiance of 20 and 70 ?mol m-2 s-1 protected the cells against damage. Cell permanence in space reduced the photosynthetic growth while the oxygen evolution capacity of the cells after the flight was enhanced. Conclusions: A potential role of PSII in capturing and utilizing ionizing radiation energy is postulated.

We have isolated the KlNOT4 gene of the yeast Kluyveromyces lactis, which encodes a component of the evolutionarily conserved CCR4-NOT complex. We show that inactivation of the gene leads to pleiotropic defects that were differentially suppressed by the NOT4 gene of S. cerevisiae, indicating that these genes have overlapping, but not identical, functions. K. lactis strains lacking Not4p are defective in fermentation and show reduced transcription of glucose transporter and glycolytic genes, which are phenotypes that are not found in the corresponding mutant of S. cerevisiae. We also show that Not4 proteins control the respiratory pathway in both yeasts, although with some differences. They activate transcription of KlACS2 and KlCYC1, but repress KlICL1, ScICL1, ScACS1, and ScCYC1. Altogether, our results indicate that Not4p is a pivotal factor involved in the regulation of carbon metabolism in yeast.

In Mycobacterium tuberculosis the decaprenyl-phospho-d-arabinofuranose (DPA) pathway is a validated target for the drugs ethambutol and benzothiazinones. To identify other potential drug targets in the pathway, we generated conditional knock-down mutants of each gene involved using the TET-PIP OFF system. dprE1, dprE2, ubiA, prsA, rv2361c, tkt and rpiB were confirmed to be essential under non-permissive conditions, whereas rv3807c was not required for survival. In the most vulnerable group, DprE1-depleted cells died faster in vitro and intracellularly than those lacking UbiA and PrsA. Downregulation of DprE1 and UbiA resulted in similar phenotypes, namely swelling of the bacteria, cell wall damage and lysis as observed at the single cell level, by real time microscopy and electron microscopy. By contrast, depletion of PrsA led to cell elongation and implosion, which was suggestive of a more pleiotropic effect. Drug sensitivity assays with known DPA-inhibitors supported the use of conditional knock-down strains for target-based whole-cell screens. Together, our work provides strong evidence for the vulnerability of all but one of the enzymes in the DPA pathway and generates valuable tools for the identification of lead compounds targeting the different biosynthetic steps. PrsA, phosphoribosyl-pyrophosphate synthetase, appears to be a particularly attractive new target for drug discovery.

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.

KlADH3 and KlADH4 are Kluyveromyces lactis genes encoding the two mitochondrial alcohol dehydrogenase activities located within mitochondria. In this yeast, ethanol induces the transcription of KlADH4 and, conversely, represses that of KlADH3. In this study, we describe the effects of the aar900 mutation on such regulation. This mutation, firstly isolated in a strain devoid of alcohol dehydrogenase genes except KlADH4, conferred to cells resistance to allyl alcohol because of the absence of the KlAdh4p activity. When the mutation was transferred by crosses to an isogenic strain containing all the alcohol dehydrogenase genes, we found that the KlADH3 gene was highly expressed even in the presence of ethanol. In addition, we observed that the absence of KlAdh4p resulted from a post-transcriptional control in that KlADH4 was transcriptionally induced by ethanol. We also found that KlPDC1, another ethanol-repressible gene, was not transcribed in the mutant in the presence of this carbon source, indicating that the escape of KlADH3 from ethanol repression was a peculiar feature of this gene. Genetic analysis showed a Mendelian segregation of the mutation that was mapped in a region of chromosome III close to the ade1 locus. Interestingly, the aar900 mutants had a pleiotropic phenotype and showed increased resistance to monovalent cations and benomyl, suggesting that the mutation could also affect genes other than the alcohol dehydrogenase ones. Strains carrying the aar900 mutation could represent useful tools to unravel the peculiar regulation of KlADH3 and KlADH4 by ethanol.

The LSM4 gene of Saccharomyces cerevisiae codes for an essential protein involved in pre-mRNA splicing and also in mRNA decapping, a crucial step for mRNA degradation. We previously demonstrated that the first 72 amino acids of the Kluyveromyces lactis Lsm4p (KlLsm4p), which contain the Sm-like domains, can restore cell viability in bothK. lactis and S. cerevisiae cells not expressing the endogenous protein. However, the absence of the carboxy-terminal region resulted in a remarkable loss of viability in stationary phase cells ( Mazzoni and Falcone, 2001 ). Herein, we demonstrate that S. cerevisiae cells expressing the truncated LSM4 protein of K. lactisshowed the phenotypic markers of yeast apoptosis such as chromatin condensation, DNA fragmentation, and accumulation of reactive oxygen species. The study of deletion mutants revealed that apoptotic markers were clearly evident also in strains lacking genes involved in mRNA decapping, such as LSM1, DCP1, andDCP2, whereas a slight effect was observed in strains lacking the genes DHH1 and PAT1. This is the first time that a connection between mRNA stability and apoptosis is reported in yeast, pointing to mRNA decapping as the crucial step responsible of the observed apoptotic phenotypes.

Reference EPFL-ARTICLE-204012doi:10.1128/Jb.02332-14View record in Web of Science Record created on 2014-12-30, modified on 2017-05-12

Summary A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr, a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme.

The emergence of multi- and extensively-drug resistant strains of Mycobacterium tuberculosis makes the development of novel anti-tubercular compounds and the identification of alternative mycobacterial drugable targets urgent priorities. Recently, type VII secretion systems (T7SS) have been discovered in mycobacteria. The genome of M. tuberculosis encodes 5 of such systems (ESX-1 to -5), three of which have been characterized and shown to be essential for viability (ESX-3, ESX-5) or virulence (ESX-1, ESX-5). Because of their crucial role in host-pathogen interactions as well as their involvement in basic biological processes of tubercle bacilli, T7SS/ESX represent promising targets for novel anti-tuberculosis drugs. Here, we review the current knowledge of the T7SS/ESX and their impact on M. tuberculosis physiology and virulence. Finally, we discuss the possible approaches to develop T7SS/ESX inhibitors.