Global analyses of TetR family transcriptional regulators in mycobacteria indicates conservation across species and diversity in regulated functions

BACKGROUND: Mycobacteria inhabit diverse niches and display high metabolic versatility. They can colonise both humans and animals and are also able to survive in the environment. In order to succeed, response to environmental cues via transcriptional regulation is required. In this study we focused on the TetR family of transcriptional regulators (TFTRs) in mycobacteria. RESULTS: We used InterPro to classify the entire complement of transcriptional regulators in 10 mycobacterial species and these analyses showed that TFTRs are the most abundant family of regulators in all species. We identified those TFTRs that are conserved across all species analysed and those that are unique to the pathogens included in the analysis. We examined genomic contexts of 663 of the conserved TFTRs and observed that the majority of TFTRs are separated by 200 bp or less from divergently oriented genes. Analyses of divergent genes indicated that the TFTRs control diverse biochemical functions not limited to efflux pumps. TFTRs typically bind to palindromic motifs and we identified 11 highly significant novel motifs in the upstream regions of divergently oriented TFTRs. The C-terminal ligand binding domain from the TFTR complement in M. tuberculosis showed great diversity in amino acid sequence but with an overall architecture common to other TFTRs. CONCLUSION: This study suggests that mycobacteria depend on TFTRs for the transcriptional control of a number of metabolic functions yet the physiological role of the majority of these regulators remain unknown. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1696-9) contains supplementary material, which is available to authorized users

Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues

Defining the pharmacological target(s) of currently used drugs and developing new analogues with greater potency are both important aspects of the search for agents that are effective against drug-sensitive and drug-resistant Mycobacterium tuberculosis. Thiacetazone (TAC) is an anti-tubercular drug that was formerly used in conjunction with isoniazid, but removed from the antitubercular chemotherapeutic arsenal due to toxic side effects. However, several recent studies have linked the mechanisms of action of TAC to mycolic acid metabolism and TAC-derived analogues have shown increased potency against M. tuberculosis. To obtain new insights into the molecular mechanisms of TAC resistance, we isolated and analyzed 10 mutants of M. tuberculosis that were highly resistant to TAC. One strain was found to be mutated in the methyltransferase MmaA4 at Gly101, consistent with its lack of oxygenated mycolic acids. All remaining strains harbored missense mutations in either HadA (at Cys61) or HadC (at Val85, Lys157 or Thr123), which are components of the bhydroxyacyl-ACP dehydratase complex that participates in the mycolic acid elongation step. Separately, a library of 31 new TAC analogues was synthesized and evaluated against M. tuberculosis. Two of these compounds, 15 and 16, exhibited minimal inhibitory concentrations 10-fold lower than the parental molecule, and inhibited mycolic acid biosynthesis in a dose-dependent manner. Moreover, overexpression of HadAB HadBC or HadABC in M. tuberculosis led to high level resistance to these compounds, demonstrating that their mode of action is similar to that of TAC. In summary, this study uncovered new mutations associated with TAC resistance and also demonstrated that simple structural optimization of the TAC scaffold was possible and may lead to a new generation of TAC-derived drug candidates for the potential treatment of tuberculosis as mycolic acid inhibitors

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment

Genome Analysis of Multi- and Extensively-Drug-Resistant Tuberculosis from KwaZulu-Natal, South Africa

The KZN strain family of Mycobacterium tuberculosis is a highly virulent strain endemic to the KwaZulu-Natal region of South Africa, which has recently experienced an outbreak of extensively-drug resistant tuberculosis. To investigate the causes and evolution of drug-resistance, we determined the DNA sequences of several clinical isolates - one drug-susceptible, one multi-drug resistant, and nine extensively drug-resistant - using whole-genome sequencing. Analysis of polymorphisms among the strains is consistent with the drug-susceptibility profiles, in that well-known mutations are observed that are correlated with resistance to isoniazid, rifampicin, kanamycin, ofloxacin, ethambutol, and pyrazinamide. However, the mutations responsible for rifampicin resistance in rpoB and pyrazinamide in pncA are in different nucleotide positions in the multi-drug-resistant and extensively drug-resistant strains, clearly showing that they acquired these mutations independently, and that the XDR strain could not have evolved directly from the MDR strain (though it could have arisen from another similar MDR strain). Sequencing of eight additional XDR strains from other areas of KwaZulu-Natal shows that they have identical drug resistant mutations to the first one sequenced, including the same polymorphisms at sites associated with drug resistance, supporting the theory that this represents a case of clonal expansion

The Binding of Triclosan to SmeT, the Repressor of the Multidrug Efflux Pump SmeDEF, Induces Antibiotic Resistance in Stenotrophomonas maltophilia

The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan

Food for pollinators: quantifying the nectar and pollen resources of urban flower meadows

Planted meadows are increasingly used to improve the biodiversity and aesthetic amenity value of urban areas. Although many ‘pollinator-friendly’ seed mixes are available, the floral resources these provide to flower-visiting insects, and how these change through time, are largely unknown. Such data are necessary to compare the resources provided by alternative meadow seed mixes to each other and to other flowering habitats. We used quantitative surveys of over 2 million flowers to estimate the nectar and pollen resources offered by two exemplar commercial seed mixes (one annual, one perennial) and associated weeds grown as 300m2 meadows across four UK cities, sampled at six time points between May and September 2013. Nectar sugar and pollen rewards per flower varied widely across 65 species surveyed, with native British weed species (including dandelion, Taraxacum agg.) contributing the top five nectar producers and two of the top ten pollen producers. Seed mix species yielding the highest rewards per flower included Leontodon hispidus, Centaurea cyanus and C. nigra for nectar, and Papaver rhoeas, Eschscholzia californica and Malva moschata for pollen. Perennial meadows produced up to 20x more nectar and up to 6x more pollen than annual meadows, which in turn produced far more than amenity grassland controls. Perennial meadows produced resources earlier in the year than annual meadows, but both seed mixes delivered very low resource levels early in the year and these were provided almost entirely by native weeds. Pollen volume per flower is well predicted statistically by floral morphology, and nectar sugar mass and pollen volume per unit area are correlated with flower counts, raising the possibility that resource levels can be estimated for species or habitats where they cannot be measured directly. Our approach does not incorporate resource quality information (for example, pollen protein or essential amino acid content), but can easily do so when suitable data exist. Our approach should inform the design of new seed mixes to ensure continuity in floral resource availability throughout the year, and to identify suitable species to fill resource gaps in established mixes