A Riboswitch-Based Inducible Gene Expression System for Mycobacteria

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

The rv1184c Locus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates

Theophylline riboswitch-controlled gene induction is reversible.

<p>(A) GFP fluorescence as a function of time in 0 mM (open) or 2 mM (filled) theophylline for <i>Msmeg</i> (circles) and <i>Mtb</i> (squares) harboring ribo-gfp. <i>Msmeg</i> vector and <i>Mtb</i> wild -type controls are shown as triangles and diamonds. Doubling times for <i>Msmeg</i> and <i>Mtb</i> are approximately 3 and 24 h, respectively. Data are presented as mean ± SEM of three independent experiments. GFP fluorescence from <i>Msmeg</i>::ribo-gfp and vector control strains was (B) monitored over time and (C) analyzed by flow cytometry after incubation with (+) or without (−) 2 mM theophylline. Theophylline was maintained or removed by media exchange after 1.3 doubling times (4 h; arrow). Kinetic data are presented as the mean ± SEM of eight replicates for each sample and are representative of three independent experiments. (D) Immunoblot analysis shows GFP induction in <i>Mtb</i> whole-cell lysates after incubation in 2 mM theophylline for one and two days (<i>top</i>). On day 2, theophylline was maintained (+) or removed by media exchange (−) and grown for an additional two days (<i>bottom</i>). Band intensities were corrected for background, and GFP signal was normalized against the GroEL loading control.</p

Theophylline induces riboswitch-controlled <i>Mtb</i> gene expression in a macrophage infection model.

<p>Murine macrophage-like RAW 264.7 cells infected with (A) <i>Mtb</i> wild type or (B) <i>Mtb</i>::ribo-gfp were induced with 0 mM or 0.5 mM theophylline for 24 h. Overlaid fluorescence signals from DAPI and GFP channels show nuclei (blue) and GFP-expressing bacteria (green). Panels on right show additional DIC light microscopy overlay. Scale bar represents 10 µm. Images are representative of three independent experiments for each condition.</p

A theophylline-responsive riboswitch variant exerts translational control of gene expression.

<p>A synthetic theophylline-responsive riboswitch variant adopts a fold that sequesters the ribosome binding site (RBS) in the mRNA transcript. In the presence of theophylline, the riboswitch adopts a conformation in which the aptamer is bound to theophylline. The RBS is then released and able to promote protein translation. (The sequence for riboswitch E′ from ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029266#pone.0029266-Topp1" target="_blank">[21]</a> is depicted.)</p

Theophylline controls endogenous KatG expression and restores sensitivity to isoniazid.

<p>(A) A single recombination event between the <i>Msmeg</i> chromosome and a plasmid containing the promoter-riboswitch combination and 500 bp of KatG yields the RiboS-<i>katG</i> strain containing a single full-length copy of <i>katG</i> under riboswitch control. The positive control for wild-type (<b>1</b>) and RiboS-katG (<b>3</b>) corresponds to the first 777 bp of <i>katG</i>. A primer specific to the promoter-riboswitch yields the predicted 1065-bp product from RiboS-<i>katG</i> (<b>4</b>), but not the wild type (<b>2</b>), confirming the recombination. (B) The isoniazid EC₅₀ for <i>Msmeg</i> wild type (open circles) and RiboS-<i>katG</i> (filled squares) was measured in response to 0–10 mM theophylline. Data are presented as mean ± SEM of three independent experiments. (<i>inset</i>) The anti-KatG immunoblot for <i>Msmeg</i> wild type and RiboS-<i>katG</i> shows the response to 0–5 mM theophylline after 6 h. The GroEL immunoblot serves as a loading control, and data are representative of two independent experiments.</p

Theophylline induces riboswitch-mediated gene expression in <i>Msmeg</i> and <i>Mtb</i>.

<p>(A) Riboswitch-controlled GFP fluorescence in <i>Msmeg</i> (filled circles) and <i>Mtb</i> (filled squares) and β-galactosidase activity in <i>Msmeg</i> (filled triangles) in response to incubation in 0–5 mM theophylline for 6 h. Empty vector negative controls for GFP fluorescence and β-galactosidase activity are shown as open circles and triangles. Data are presented as relative fluorescence (RFU) for GFP and in Miller units for β-galactosidase, and as the mean ± SEM of three independent experiments. (B) Flow cytometry analysis of riboswitch-controlled GFP expression in <i>Msmeg</i> treated for 6 h with varying concentrations of theophylline. The empty vector control is shown in black. Results are representative of three or more independent experiments. (C) Immunoblot analysis of whole-cell lysates from <i>Mtb</i> harboring ribo-gfp, empty vector, or Phsp60-gfp positive control constructs. Band intensities were corrected for background, and GFP signal was normalized against the GroEL loading control.</p