Publications by authors named "Amanda C Brown"

21 Publications

  • Page 1 of 1

The Deconstructed Granuloma: A Complex High-Throughput Drug Screening Platform for the Discovery of Host-Directed Therapeutics Against Tuberculosis.

Front Cell Infect Microbiol 2018 14;8:275. Epub 2018 Aug 14.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States.

(Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone. The absence of these players unnecessarily limits the breadth of novel host target pathways to be interrogated. In this study, we detail a new drug screening platform based on dissociated murine TB granulomas, named the Deconstructed Granuloma (DGr), that utilizes fluorescent Mtb reporter strains screened in the host immune environment of the infection site. The platform has been used to screen a collection of known drug candidates. Data from a representative 384-well plate containing known anti-bacterial compounds are shown, illustrating the robustness of the screening platform. The novel deconstructed granuloma platform represents an accessible, sensitive and robust high-throughput screen suitable for the inclusive interrogation of immune targets for Host-Directed Therapeutics.
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http://dx.doi.org/10.3389/fcimb.2018.00275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102409PMC
August 2019

Micrococcin P1 - A bactericidal thiopeptide active against Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2016 09 27;100:95-101. Epub 2016 Jul 27.

Department of Molecular Medicine, University of Padova, Padova, Italy. Electronic address:

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.
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http://dx.doi.org/10.1016/j.tube.2016.07.011DOI Listing
September 2016

Islands of linkage in an ocean of pervasive recombination reveals two-speed evolution of human cytomegalovirus genomes.

Virus Evol 2016 Jan 15;2(1):vew017. Epub 2016 Jun 15.

Division of Infection and Immunity, University College London, London, United Kingdom.

Human cytomegalovirus (HCMV) infects most of the population worldwide, persisting throughout the host's life in a latent state with periodic episodes of reactivation. While typically asymptomatic, HCMV can cause fatal disease among congenitally infected infants and immunocompromised patients. These clinical issues are compounded by the emergence of antiviral resistance and the absence of an effective vaccine, the development of which is likely complicated by the numerous immune evasins encoded by HCMV to counter the host's adaptive immune responses, a feature that facilitates frequent super-infections. Understanding the evolutionary dynamics of HCMV is essential for the development of effective new drugs and vaccines. By comparing viral genomes from uncultivated or low-passaged clinical samples of diverse origins, we observe evidence of frequent homologous recombination events, both recent and ancient, and no structure of HCMV genetic diversity at the whole-genome scale. Analysis of individual gene-scale loci reveals a striking dichotomy: while most of the genome is highly conserved, recombines essentially freely and has evolved under purifying selection, 21 genes display extreme diversity, structured into distinct genotypes that do not recombine with each other. Most of these hyper-variable genes encode glycoproteins involved in cell entry or escape of host immunity. Evidence that half of them have diverged through episodes of intense positive selection suggests that rapid evolution of hyper-variable loci is likely driven by interactions with host immunity. It appears that this process is enabled by recombination unlinking hyper-variable loci from strongly constrained neighboring sites. It is conceivable that viral mechanisms facilitating super-infection have evolved to promote recombination between diverged genotypes, allowing the virus to continuously diversify at key loci to escape immune detection, while maintaining a genome optimally adapted to its asymptomatic infectious lifecycle.
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http://dx.doi.org/10.1093/ve/vew017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167919PMC
January 2016

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

J Clin Microbiol 2015 Jul 13;53(7):2230-7. Epub 2015 May 13.

UCL, Division of Infection and Immunity, London, United Kingdom.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.
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http://dx.doi.org/10.1128/JCM.00486-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473240PMC
July 2015

Mycobacterium tuberculosis H37Rv has a single nucleotide polymorphism in PhoR which affects cell wall hydrophobicity and gene expression.

Microbiology (Reading) 2015 Apr 29;161(Pt 4):765-773. Epub 2015 Jan 29.

2TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA.

Mycobacterium tuberculosis is a successful pathogen that can adapt to multiple environmental niches. As part of its repertoire of adaptive responses, two-component regulatory systems play a major role in co-ordinating gene expression at the global level. The PhoPR system controls major cellular functions, including respiration, lipid metabolism, the immediate and enduring hypoxic responses, stress responses and persistence. We identified a single nucleotide polymorphism (SNP) found in the sensor kinase (PhoR) of this system between two commonly used strains of M. tuberculosis, H37Rv (PhoR(P152)) and CDC1551 (PhoR(L152)). We constructed an isogenic strain of H37Rv carrying PhoR(L152), as well as strains containing two different copies of the PhoPR locus, to determine the functional consequences of the SNP on phenotypic traits. The previously identified Apr locus was not acid-inducible in H37Rv, although it was in the CDC1551 strain. Surprisingly, the acid-responsive expression was not completely dependent on the PhoR SNP, and the locus remained constitutively expressed even in the isogenic strain H37Rv:PhoR(L152). The pattern of expression in PhoPR merodiploid strains was more complex, with neither allele showing dominance. This suggests that Apr regulation is more complex than previously thought and that additional factors must be responsible for Apr upregulation in response to acid conditions. In contrast, differences we identified in cell hydrophobicity between the two strains were wholly dependent on PhoR, confirming its role as major regulator of cell wall composition. Thus the SNP in the sensor kinase has functional consequences which account for some of the differences between widely used laboratory strains.
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http://dx.doi.org/10.1099/mic.0.000036DOI Listing
April 2015

Whole-genome enrichment and sequencing of Chlamydia trachomatis directly from clinical samples.

BMC Infect Dis 2014 Nov 12;14:591. Epub 2014 Nov 12.

Division of Infection and Immunity University College London (UCL), London, WC1E 6BT, UK.

Background: Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples.

Methods: C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform.

Results: Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies.

Conclusions: The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
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http://dx.doi.org/10.1186/s12879-014-0591-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233057PMC
November 2014

Identification of the likely translational start of Mycobacterium tuberculosis GyrB.

BMC Res Notes 2013 Jul 15;6:274. Epub 2013 Jul 15.

Department of Biological Chemistry, John Innes Centre Norwich Research Park, Norwich NR4 7UH, UK.

Background: Bacterial DNA gyrase is a validated target for antibacterial chemotherapy. It consists of two subunits, GyrA and GyrB, which form an A₂B₂ complex in the active enzyme. Sequence alignment of Mycobacterium tuberculosis GyrB with other bacterial GyrBs predicts the presence of 40 potential additional amino acids at the GyrB N-terminus. There are discrepancies between the M. tuberculosis GyrB sequences retrieved from different databases, including sequences annotated with or without the additional 40 amino acids. This has resulted in differences in the GyrB sequence numbering that has led to the reporting of previously known fluoroquinolone-resistance mutations as novel mutations.

Findings: We have expressed M. tuberculosis GyrB with and without the extra 40 amino acids in Escherichia coli and shown that both can be produced as soluble, active proteins. Supercoiling and other assays of the two proteins show no differences, suggesting that the additional 40 amino acids have no effect on the enzyme in vitro. RT-PCR analysis of M. tuberculosis mRNA shows that transcripts that could yield both the longer and shorter protein are present. However, promoter analysis showed that only the promoter elements leading to the shorter GyrB (lacking the additional 40 amino acids) had significant activity.

Conclusion: We conclude that the most probable translational start codon for M. tuberculosis GyrB is GTG (Val) which results in translation of a protein of 674 amino acids (74 kDa).
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http://dx.doi.org/10.1186/1756-0500-6-274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724585PMC
July 2013

Mycobacterium tuberculosis ClpP proteases are co-transcribed but exhibit different substrate specificities.

PLoS One 2013 1;8(4):e60228. Epub 2013 Apr 1.

Queen Mary University of London, Barts & The London School of Medicine and Dentistry, London E1 2AT, United Kingdom.

Caseinolytic (Clp) proteases are widespread energy-dependent proteases; the functional ATP-dependent protease is comprised of multimers of proteolytic and regulatory subunits. Mycobacterium tuberculosis has two ClpP proteolytic subunits (ClpP1 and ClpP2), with both being essential for growth in vitro. ClpP1 and clpP2 are arranged in an apparent operon; we demonstrated that the two genes are co-expressed under normal growth conditions. We identified a single promoter region for the clpP1P2 operon; no promoter was detected upstream of clpP2 demonstrating that independent expression of clpP1 and clpP2 was highly unlikely. Promoter activity was not induced by heat shock or oxidative stress. We identified a regulatory region upstream of the promoter with a consensus sequence matching the ClgR regulator motif; we determined the limits of the region by mutagenesis and confirmed that positive regulation of the promoter occurs in M. tuberculosis. We developed a reporter system to monitor ClpP1 and ClpP2 enzymatic activities based on LacZ incorporating ssrAtag sequences. We showed that whilst both ClpP1 and ClpP2 degrade SsrA-tagged LacZ, ClpP2 (but not ClpP1) degrades untagged proteins. Our data suggest that the two proteolytic subunits display different substrate specificities and therefore have different, but overlapping roles in M. tuberculosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060228PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613350PMC
September 2013

Deletion of SenX3-RegX3, a key two-component regulatory system of Mycobacterium smegmatis, results in growth defects under phosphate-limiting conditions.

Microbiology (Reading) 2012 Nov 6;158(Pt 11):2724-2731. Epub 2012 Sep 6.

TB Discovery Research, Infectious Disease Research Institute, Seattle, WA, USA.

Two component regulatory systems are key elements in the control of bacterial gene expression in response to environmental perturbations. The SenX3-RegX3 system is implicated in the control of phosphate uptake in Mycobacterium smegmatis and Mycobacterium tuberculosis. regX3 is reported to be essential in M. smegmatis, but not in M. tuberculosis. We attempted to construct complete senX3-regX3 operon deletion strains of M. smegmatis; initially we found that the operon could only be deleted when another functional copy was provided. Using a strain in which the only functional copy of the operon was present on an integrating plasmid, we attempted to replace the functional copy with an empty vector. Surprisingly, we obtained strains in which the functional copy had been deleted from the chromosome at a low frequency. We deleted the senX3 gene in a similar fashion, but it was not possible to delete regX3 alone. To identify possible compensatory mutations we sequenced the whole genome of two deletion strains and the wild-type. A synonymous single nucleotide polymorphism (SNP) in a lipoprotein was found in all deletion strains, but not the parental strains, and a frameshift mutation in nhaA was identified in three of the four deletion strains. Operon deletion strains were more sensitive to phosphate limitation, showing a reduced ability to grow at lower phosphate concentrations. The M. tuberculosis operon was able to functionally complement the growth phenotype in M. smegmatis under phosphate-replete conditions, but not under low phosphate conditions, reinforcing the difference between the two species. Our data show that, in contrast with previous reports, it is possible to delete the operon in M. smegmatis, possibly due to the accumulation of compensatory mutations, and that the deletion does affect growth in phosphate.
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http://dx.doi.org/10.1099/mic.0.060319-0DOI Listing
November 2012

The dUTPase enzyme is essential in Mycobacterium smegmatis.

PLoS One 2012 24;7(5):e37461. Epub 2012 May 24.

Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest, Hungary.

Thymidine biosynthesis is essential in all cells. Inhibitors of the enzymes involved in this pathway (e.g. methotrexate) are thus frequently used as cytostatics. Due to its pivotal role in mycobacterial thymidylate synthesis dUTPase, which hydrolyzes dUTP into the dTTP precursor dUMP, has been suggested as a target for new antitubercular agents. All mycobacterial genomes encode dUTPase with a mycobacteria-specific surface loop absent in the human dUTPase. Using Mycobacterium smegmatis as a fast growing model for Mycobacterium tuberculosis, we demonstrate that dUTPase knock-out results in lethality that can be reverted by complementation with wild-type dUTPase. Interestingly, a mutant dUTPase gene lacking the genus-specific loop was unable to complement the knock-out phenotype. We also show that deletion of the mycobacteria-specific loop has no major effect on dUTPase enzymatic properties in vitro and thus a yet to be identified loop-specific function seems to be essential within the bacterial cell context. In addition, here we demonstrated that Mycobacterium tuberculosis dUTPase is fully functional in Mycobacterium smegmatis as it rescues the lethal knock-out phenotype. Our results indicate the potential of dUTPase as a target for antitubercular drugs and identify a genus-specific surface loop on the enzyme as a selective target.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037461PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360063PMC
September 2012

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection.

PLoS Pathog 2011 May 12;7(5):e1002040. Epub 2011 May 12.

Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff, United Kingdom.

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.
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http://dx.doi.org/10.1371/journal.ppat.1002040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093373PMC
May 2011

The nonmevalonate pathway of isoprenoid biosynthesis in Mycobacterium tuberculosis is essential and transcriptionally regulated by Dxs.

J Bacteriol 2010 May 19;192(9):2424-33. Epub 2010 Feb 19.

Queen Mary University of London, Barts and the London School of Medicine and Dentistry, London E12AD, United Kingdom.

Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator.
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http://dx.doi.org/10.1128/JB.01402-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863480PMC
May 2010

Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens.

Chem Biol 2009 Dec;16(12):1230-9

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.

Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
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http://dx.doi.org/10.1016/j.chembiol.2009.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020808PMC
December 2009

Dxr is essential in Mycobacterium tuberculosis and fosmidomycin resistance is due to a lack of uptake.

BMC Microbiol 2008 May 20;8:78. Epub 2008 May 20.

Institute for Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK.

Unlabelled: Fosmidomycin is a phosphonic antibiotic which inhibits 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), the first committed step of the non-mevalonate pathway of isoprenoid biosynthesis. In Mycobacterium tuberculosis Dxr is encoded by Rv2870c, and although the antibiotic has been shown to inhibit the recombinant enzyme 1, mycobacteria are intrinsically resistant to fosmidomycin at the whole cell level. Fosmidomycin is a hydrophilic molecule and in many bacteria its uptake is an active process involving a cAMP dependent glycerol-3-phosphate transporter (GlpT). The fact that there is no glpT homologue in the M. tuberculosis genome and the highly impervious nature of the hydrophobic mycobacterial cell wall suggests that resistance may be due to a lack of cellular penetration.

Results: We demonstrated that dxr (Rv2780c) is an essential gene in M. tuberculosis, since we could not delete the chromosomal copy unless a second functional copy was provided on an integrating vector. This confirmed that the intracellular target of fosmidomycin was essential as well as sensitive. We looked at the uptake of fosmidomycin in two mycobacterial species, the slow-growing pathogenic M. tuberculosis and the fast-growing, saprophytic Mycobacterium smegmatis; both species were resistant to fosmidomycin to a high level. Fosmidomycin was not accumulated intra-cellularly in M. tuberculosis or M. smegmatis but remained in the extra-cellular medium. In contrast, fosmidomycin uptake was confirmed in the sensitive organism, Escherichia coli. We established that the lack of intra-cellular accumulation was not due to efflux, since efflux pump inhibitors had no effect on fosmidomycin resistance. Finally, we demonstrated that fosmidomycin was not modified by mycobacterial cells or by extracts but remained in a fully functional state.

Conclusion: Taken together, these data demonstrate that fosmidomycin resistance in M. tuberculosis and M. smegmatis results from a lack of penetration of the antibiotic to the site of the sensitive target.
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http://dx.doi.org/10.1186/1471-2180-8-78DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2409342PMC
May 2008

The structure of Mycobacteria 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase, an essential enzyme, provides a platform for drug discovery.

BMC Struct Biol 2007 Oct 23;7:68. Epub 2007 Oct 23.

Division of Biological Chemistry and Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

Background: The prevalence of tuberculosis, the prolonged and expensive treatment that this disease requires and an increase in drug resistance indicate an urgent need for new treatments. The 1-deoxy-D-xylulose 5-phosphate pathway of isoprenoid precursor biosynthesis is an attractive chemotherapeutic target because it occurs in many pathogens, including Mycobacterium tuberculosis, and is absent from humans. To underpin future drug development it is important to assess which enzymes in this biosynthetic pathway are essential in the actual pathogens and to characterize them.

Results: The fifth enzyme of this pathway, encoded by ispF, is 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF). A two-step recombination strategy was used to construct ispF deletion mutants in M. tuberculosis but only wild-type double crossover strains were isolated. The chromosomal copy could be deleted when a second functional copy was provided on an integrating plasmid, demonstrating that ispF is an essential gene under the conditions tested thereby confirming its potential as a drug target. We attempted structure determination of the M. tuberculosis enzyme (MtIspF), but failed to obtain crystals. We instead analyzed the orthologue M. smegmatis IspF (MsIspF), sharing 73% amino acid sequence identity, at 2.2 A resolution. The high level of sequence conservation is particularly pronounced in and around the active site. MsIspF is a trimer with a hydrophobic cavity at its center that contains density consistent with diphosphate-containing isoprenoids. The active site, created by two subunits, comprises a rigid CDP-Zn2+ binding pocket with a flexible loop to position the 2C-methyl-D-erythritol moiety of substrate. Sequence-structure comparisons indicate that the active site and interactions with ligands are highly conserved.

Conclusion: Our study genetically validates MtIspF as a therapeutic target and provides a model system for structure-based ligand design.
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http://dx.doi.org/10.1186/1472-6807-7-68DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151065PMC
October 2007

Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase: potential for drug development.

J Bacteriol 2007 Dec 5;189(24):8922-7. Epub 2007 Oct 5.

Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.

Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 microM for MEP and 53.2 microM for CTP. Calculated kcat and kcat/Km values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.
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http://dx.doi.org/10.1128/JB.00925-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168624PMC
December 2007

Expression of Mycobacterium tuberculosis Rv1991c using an arabinose-inducible promoter demonstrates its role as a toxin.

FEMS Microbiol Lett 2007 Sep 9;274(1):73-82. Epub 2007 Jul 9.

Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, Whitechapel, London, UK.

Conditional gene expression systems are useful tools for studying the role of essential or toxic gene products in bacterial systems. There is a paucity of such systems available for use in the mycobacteria. The utility of the Escherichia coli arabinose-inducible system was looked into, since it is tightly controlled in response to the presence of arabinose and glucose. It was demonstrated that the P(BAD) promoter can be used to express heterologous genes in Mycobacterium smegmatis. Expression of a lacZ reporter gene demonstrated that promoter activity was inducible in response to the presence of glucose, but only on solid medium. This system was utilized to study the functional consequences of expressing one member of a putative toxin-antitoxin pair (Rv1991c). Rv1991c has homology with a number of bacterial toxins, including ChpK, MazF and PemK. A potential antitoxin gene has been identified, adjacent to Rv1991c in the genome, which was coexpressed with the toxin. Expression of the toxin alone inhibited the growth of E. coli, whereas coexpression with the antitoxin did not. Expression of Rv1991c also led to a marked reduction of cell viability in M. smegmatis, confirming its role as a potent toxin.
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http://dx.doi.org/10.1111/j.1574-6968.2007.00842.xDOI Listing
September 2007

Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability.

Microbiology (Reading) 2006 Sep;152(Pt 9):2727-2734

Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and the London, London E1 2AT, UK.

Adenylyltransferase, GlnE, has a predicted role in controlling the enzymic activity of glutamine synthetase, the key enzyme in ammonia assimilation. It was previously demonstrated that glnE is an essential gene in Mycobacterium tuberculosis. glnE is located downstream of glnA2, one of four glutamine synthetases. The expression of GlnE under various conditions was determined. Although a co-transcript of glnA2 and glnE was detectable, the major transcript was monocistronic. A transcriptional start site immediately upstream of glnE was identified and it was shown by site-directed mutagenesis that the predicted -10 region is a functional promoter. It was demonstrated that in a Mycobacterium smegmatis background M. tuberculosis P(glnE) was up-regulated in ammonia- or glutamine-containing media.
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http://dx.doi.org/10.1099/mic.0.28942-0DOI Listing
September 2006

Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis.

Plasmid 2006 Jan 24;55(1):81-6. Epub 2005 Aug 24.

Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and the London, Queen Mary's School of Medicine and Dentistry, Turner Street, London E1 2AD, UK.

The Escherichia coli-mycobacterium shuttle vector pJAM2 has been used to inducibly express genes in mycobacteria. The vector carries the promoter region from the highly inducible acetamidase gene of Mycobacterium smegmatis which is used to drive expression of heterologous genes. We used pJAM2 to over-express the Mycobacterium tuberculosis gene Rv2868c, a homologue of gcpE. In M. smegmatis the plasmid was stable, but the promoter region was readily deleted when the parental vector or recombinant plasmids were transformed into M. tuberculosis. We mapped the deletion by sequencing and found that it encompassed the entire acetamidase promoter and adjacent sequence totalling approximately 7.3 kb and occurred very soon after introduction into M. tuberculosis. This is the first report of instability of a vector carrying the acetamidase promoter in M. tuberculosis.
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http://dx.doi.org/10.1016/j.plasmid.2005.06.005DOI Listing
January 2006

Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b.

Eur J Biochem 2004 Jun;271(11):2264-71

The Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK.

Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Q(o) inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases. Specific residues in the Q(o) site of yeast cytochrome b were modified to obtain four new forms mimicking the Q(o) binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma. These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc(1) complex activity. In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied. The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin. The pattern of resistance conferred by F129L and L275F was different. Interestingly G143A had a slightly deleterious effect on the bc(1) function in V. inaequalis, S. fuliginea and P. megasperma Q(o) site mimics but not in that for E. graminis. Thus small variations in the Q(o) site seem to affect the impact of the G143A mutation on bc(1) activity. Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially. If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens.
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http://dx.doi.org/10.1111/j.1432-1033.2004.04169.xDOI Listing
June 2004

Characterisation of target-site resistance to ACCase-inhibiting herbicides in the weed Alopecurus myosuroides (black-grass).

Pest Manag Sci 2003 Feb;59(2):190-201

Division of Plant and Invertebrate Ecology, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK.

Resistance to aryloxyphenoxypropionate (AOPP), cyclohexanedione (CHD) and phenylurea herbicides was determined in UK populations of Alopecurus myosuroides Huds. Two populations (Oxford AA1, Notts. A1) were highly resistant (Resistance indices 13-->1000) to the AOPP and CHD herbicides fenoxaprop, diclofop, fluazifop-P and sethoxydim, but only marginally resistant to the phenylurea, chlorotoluron. Analyses of acetyl coenzyme A carboxylase (ACCase) activity showed that an insensitive ACCase conferred resistance to all the AOPP/CHD herbicides investigated. Another population, Oxford S1, showed no resistance to sethoxydim at the population level, but contained a small proportion of plants (<10%) with an insensitive ACCase. Genetic studies on the Notts A1 and Oxford S1 populations demonstrated that target site resistance conferred by an insensitive ACCase is monogenic, nuclearly inherited with the resistant allele showing complete dominance. Investigations of the molecular basis of resistance in the Notts A1 population showed that sethoxydim resistance in A myosuroides was associated with the substitution of an isoleucine in susceptible with a leucine in resistant plants, which has also been found in three other resistant grass-weed species (Setaria viridis (L) Beauv, Avena fatua L, Lolium rigidum Gaud).
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http://dx.doi.org/10.1002/ps.623DOI Listing
February 2003