A temperature-sensitive Mycobacterium smegmatis glgE mutation leads to a loss of GlgE enzyme activity and thermostability and the accumulation of α-maltose-1-phosphate

Background: The bacterial GlgE pathway is the third known route to glycogen and is the only one present in mycobacteria. It contributes to the virulence of Mycobacterium tuberculosis. The involvement of GlgE in glycogen biosynthesis was discovered twenty years ago when the phenotype of a temperature-sensitive Mycobacterium smegmatis mutation was rescued by the glgE gene. The evidence at the time suggested glgE coded for a glucanase responsible for the hydrolysis of glycogen, in stark contrast with recent evidence showing GlgE to be a polymerase responsible for its biosynthesis. Methods: We reconstructed and examined the temperature-sensitive mutant and characterised the mutated GlgE enzyme. Results: The mutant strain accumulated the substrate for GlgE, α-maltose-1-phosphate, at the non-permissive temperature. The glycogen assay used in the original study was shown to give a false positive result with α-maltose-1-phosphate. The accumulation of α-maltose-1-phosphate was due to the lowering of the kcat of GlgE as well as a loss of stability 42 °C. The reported rescue of the phenotype by GarA could potentially involve an interaction with GlgE, but none was detected. Conclusions: We have been able to reconcile apparently contradictory observations and shed light on the basis for the phenotype of the temperature-sensitive mutation. General significance: This study highlights how the lowering of flux through the GlgE pathway can slow the growth mycobacteria

Metabolic Network for the Biosynthesis of Intra- and Extracellular alpha-Glucans Required for Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis synthesizes intra- and extracellular alpha-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the alpha-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of alpha-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in alpha-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that alpha-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block alpha-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some alpha-glucan is exported to form the alpha-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate alpha-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADPglucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying alpha-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of alpha-glucan could be constructed, showing a direct link between the GlgE pathway, alpha-glucan biosynthesis and virulence in a mouse infection model

Assembly of α-Glucan by GlgE and GlgB in Mycobacteria and Streptomycetes

Actinomycetes, such as mycobacteria and streptomycetes, synthesize α-glucan with α-1,4 linkages and α-1,6 branching to help evade immune responses and to store carbon. α-Glucan is thought to resemble glycogen except for having shorter constituent linear chains. However, the fine structure of α-glucan and how it can be defined by the maltosyl transferase GlgE and branching enzyme GlgB were not known. Using a combination of enzymolysis and mass spectrometry, we compared the properties of α-glucan isolated from actinomycetes with polymer synthesized in vitro by GlgE and GlgB. We now propose the following assembly mechanism. Polymer synthesis starts with GlgE and its donor substrate, α-maltose 1-phosphate, yielding a linear oligomer with a degree of polymerization (∼16) sufficient for GlgB to introduce a branch. Branching involves strictly intrachain transfer to generate a C chain (the only constituent chain to retain its reducing end), which now bears an A chain (a nonreducing end terminal branch that does not itself bear a branch). GlgE preferentially extends A chains allowing GlgB to act iteratively to generate new A chains emanating from B chains (nonterminal branches that themselves bear a branch). Although extension and branching occur primarily with A chains, the other chain types are sometimes extended and branched such that some B chains (and possibly C chains) bear more than one branch. This occurs less frequently in α-glucans than in classical glycogens. The very similar properties of cytosolic and capsular α-glucans from Mycobacterium tuberculosis imply GlgE and GlgB are sufficient to synthesize them both

A Search for TeV Gamma-Ray Emission from High-Peaked Flat Spectrum Radio Quasars Using the Whipple Air-Cherenkov Telescope

Blazars have traditionally been separated into two broad categories based upon their optical emission characteristics; BL Lacs, with faint or no emission lines, and flat spectrum radio quasars (FSRQs) with prominent, broad emission lines. The spectral energy distribution of FSRQs has generally been thought of as being more akin to the low-peaked BL Lacs, which exhibit a peak in the infrared region of the spectrum, as opposed to high-peaked BL Lacs (HBLs), which exhibit a peak in UV/X-ray region of the spectrum. All blazars currently confirmed as sources of TeV emission are HBLs. Recent surveys have found several FSRQs exhibiting spectral properties similar to HBLs, particularly the synchrotron peak frequency. These objects are potential sources of TeV emission according to several models of blazar jet emission and blazar evolution. Measurements of TeV flux or upper limits could impact existing theories explaining the links between different blazar types and could have a significant impact on our understanding of the nature of objects that are capable of TeV emission. In particular, the presence (or absence) of TeV emission from FSRQs could confirm (or cast doubt upon) recent evolutionary models that expect intermediate objects in a transitionary state between FSRQ and BL Lac. The Whipple 10 meter imaging air-Cherenkov gamma-ray telescope is well suited for TeV gamma-ray observations. Using the Whipple telescope, we have taken data on a small selection of nearby(z<0.1 in most cases), high-peaked FSRQs. Although one of the objects, B2 0321+33, showed marginal evidence of flaring, no significant emission was detected. The implications of this paucity of emission and the derived upper limits are discussed.Comment: accepted for publication in Astrophysical Journa

The First VERITAS Telescope

The first atmospheric Cherenkov telescope of VERITAS (the Very Energetic Radiation Imaging Telescope Array System) has been in operation since February 2005. We present here a technical description of the instrument and a summary of its performance. The calibration methods are described, along with the results of Monte Carlo simulations of the telescope and comparisons between real and simulated data. The analysis of TeV $\gamma$-ray observations of the Crab Nebula, including the reconstructed energy spectrum, is shown to give results consistent with earlier measurements. The telescope is operating as expected and has met or exceeded all design specifications.Comment: Accepted by Astroparticle Physic

A Multi-wavelength View of the TeV Blazar Markarian 421: Correlated Variability, Flaring, and Spectral Evolution

We report results from a multi-wavelength monitoring campaign on Mrk 421 over the period of 2003-2004. The source was observed simultaneously at TeV and X-ray energies, with supporting observations frequently carried out at optical and radio wavelengths. The large amount of simultaneous data has allowed us to examine the variability of Mrk 421 in detail. The variabilities are generally correlated between the X-ray and gamma-ray bands, although the correlation appears to be fairly loose. The light curves show the presence of flares with varying amplitudes on a wide range of timescales both at X-ray and TeV energies. Of particular interest is the presence of TeV flares that have no coincident counterparts at longer wavelengths, because the phenomenon seems difficult to understand in the context of the proposed emission models for TeV blazars. We have also found that the TeV flux reached its peak days before the X-ray flux during a giant flare in 2004. Such a difference in the development of the flare presents a further challenge to the emission models. Mrk 421 varied much less at optical and radio wavelengths. Surprisingly, the normalized variability amplitude in optical seems to be comparable to that in radio, perhaps suggesting the presence of different populations of emitting electrons in the jet. The spectral energy distribution (SED) of Mrk 421 is seen to vary with flux, with the two characteristic peaks moving toward higher energies at higher fluxes. We have failed to fit the measured SEDs with a one-zone SSC model; introducing additional zones greatly improves the fits. We have derived constraints on the physical properties of the X-ray/gamma-ray flaring regions from the observed variability (and SED) of the source. The implications of the results are discussed. (Abridged)Comment: 32 pages, 12 figures, to appear in Ap

Studying the association between musculoskeletal disorders, quality of life and mental health. A primary care pilot study in rural Crete, Greece

<p>Abstract</p> <p>Background</p> <p>The burden of musculoskeletal disorders (MSD) on the general health and well-being of the population has been documented in various studies. The objective of this study was to explore the association between MSD and the quality of life and mental health of patients and to discuss issues concerning care seeking patterns in rural Greece.</p> <p>Methods</p> <p>Patients registered at one rural Primary Care Centre (PCC) in Crete were invited to complete the Nordic Musculoskeletal Questionnaire (NMQ) for the analysis of musculoskeletal symptoms, together with validated instruments for measuring health related quality of life (SF-36) and mental distress (GHQ-28).</p> <p>Results</p> <p>The prevalence rate of MSD was found to be 71.2%, with low back and knee pain being the most common symptoms. Most conditions significantly impaired the quality of life, especially the physical dimensions of SF-36. Depression was strongly correlated to most MSD (<it>p </it>< 0.001). Multiple logistic analyses revealed that patients who consulted the PCC due to MSD were likely to have more mental distress or impaired physical functioning compared to those who did not.</p> <p>Conclusion</p> <p>Musculoskeletal disorders were common in patients attending the rural PCC of this study and were associated with a poor quality of life and mental distress that affected their consultation behaviour.</p

Trehalose-6-phosphate-mediated toxicity determines essentiality of OtsB2 in Mycobacterium tuberculosis in vitro and in mice

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation

Lifestyle intervention in obese pregnancy and cardiac remodelling in 3-year olds: children of the UPBEAT RCT

Background/Objectives: Obesity in pregnancy has been associated with increased childhood cardiometabolic risk and reduced life expectancy. The UK UPBEAT multicentre randomised control trial was a lifestyle intervention of diet and physical activity in pregnant women with obesity. We hypothesised that the 3-year-old children of women with obesity would have heightened cardiovascular risk compared to children of normal BMI women, and that the UPBEAT intervention would mitigate this risk. Subjects/Methods: Children were recruited from one UPBEAT trial centre. Cardiovascular measures included blood pressure, echocardiographic assessment of cardiac function and dimensions, carotid intima-media thickness and heart rate variability (HRV) by electrocardiogram. Results: Compared to offspring of normal BMI women (n = 51), children of women with obesity from the trial standard care arm (n = 39) had evidence of cardiac remodelling including increased interventricular septum (IVS; mean difference 0.04 cm; 95% CI: 0.018 to 0.067), posterior wall (PW; 0.03 cm; 0.006 to 0.062) and relative wall thicknesses (RWT; 0.03 cm; 0.01 to 0.05) following adjustment. Randomisation of women with obesity to the intervention arm (n = 31) prevented this cardiac remodelling (intervention effect; mean difference IVS −0.03 cm (−0.05 to −0.008); PW −0.03 cm (−0.05 to −0.01); RWT −0.02 cm (−0.04 to −0.005)). Children of women with obesity (standard care arm) compared to women of normal BMI also had elevated minimum heart rate (7 bpm; 1.41 to 13.34) evidence of early diastolic dysfunction (e prime) and increased sympathetic nerve activity index by HRV analysis. Conclusions: Maternal obesity was associated with left ventricular concentric remodelling in 3-year-old offspring. Absence of remodelling following the maternal intervention infers in utero origins of cardiac remodelling. Clinical trial registry name and registration number: The UPBEAT trial is registered with Current Controlled Trials, ISRCTN89971375