Publications
130 results found
Andreu N, Fletcher T, Krishnan N, et al., 2011, Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence, Journal of Antimicrobial Chemotherapy, Vol: 67, Pages: 404-414, ISSN: 1460-2091
Objectives Tuberculosis drug development is hampered by the slow growth of Mycobacterium tuberculosis. Bioluminescence, light produced by an enzymatic reaction, constitutes a rapid and highly sensitive measurement of cell metabolic function that can be used as an indirect marker of cell viability in drug screening assays. The aim of this work was to validate and standardize the use of luminescent M. tuberculosis strains to test the activity of antibacterial drugs in vitro and inside macrophages in a 96-well format.Methods We have used strains that express the bacterial lux operon and therefore do not require exogenous substrate to produce light, as well as strains expressing the firefly luciferase that need luciferin substrate. Results were compared with those obtained using the resazurin reduction assay and cfu plating.Results Using bioluminescence we were able to reduce the time required to measure the MIC and bactericidal concentrations of antimicrobials to just 3 and 6 days, respectively. Furthermore, antibacterial activity against intracellular mycobacteria was detected within 2 days post-infection. Results were comparable to those obtained by conventional methods.Conclusions We have developed a simple and rapid method for screening antimycobacterial drugs in culture and in macrophages. The use of autoluminescent bacteria also facilitates the determination of growth and inhibition kinetics. The method is cost-effective, can easily be adapted to a larger scale and is amenable to automation. Current efforts are directed towards applying this technology to drug screening in vivo.
Williams KJ, Boshoff HI, Krishnan N, et al., 2011, The <i>Mycobacterium tuberculosis</i> β-oxidation genes <i>echA5</i> and <i>fadB3</i> are dispensable for growth <i>in vitro</i> and <i>in vivo</i>, TUBERCULOSIS, Vol: 91, Pages: 549-555, ISSN: 1472-9792
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- Citations: 23
Krishnan N, Malaga W, Constant P, et al., 2011, Mycobacterium tuberculosis Lineage Influences Innate Immune Response and Virulence and Is Associated with Distinct Cell Envelope Lipid Profiles, PLOS One, Vol: 6, ISSN: 1932-6203
The six major genetic lineages of Mycobacterium tuberculosis are strongly associated with specific geographical regions, but their relevance to bacterial virulence and the clinical consequences of infection are unclear. Previously, we found that in Vietnam, East Asian/Beijing and Indo-Oceanic strains were significantly more likely to cause disseminated tuberculosis with meningitis than those from the Euro-American lineage. To investigate this observation we characterised 7 East Asian/Beijing, 5 Indo-Oceanic and 6 Euro-American Vietnamese strains in bone-marrow-derived macrophages, dendritic cells and mice. East Asian/Beijing and Indo-Oceanic strains induced significantly more TNF-α and IL-1β from macrophages than the Euro-American strains, and East Asian/Beijing strains were detectable earlier in the blood of infected mice and grew faster in the lungs. We hypothesised that these differences were induced by lineage-specific variation in cell envelope lipids. Whole lipid extracts from East Asian/Beijing and Indo-Oceanic strains induced higher concentrations of TNF-α from macrophages than Euro-American lipids. The lipid extracts were fractionated and compared by thin layer chromatography to reveal a distinct pattern of lineage-associated profiles. A phthiotriol dimycocerosate was exclusively produced by East Asian/Beijing strains, but not the phenolic glycolipid previously associated with the hyper-virulent phenotype of some isolates of this lineage. All Indo-Oceanic strains produced a unique unidentified lipid, shown to be a phenolphthiocerol dimycocerosate dependent upon an intact pks15/1 for its production. This was described by Goren as the ‘attenuation indictor lipid’ more than 40 years ago, due to its association with less virulent strains from southern India. Mutation of pks15/1 in a representative Indo-Oceanic strain prevented phenolphthiocerol dimycocerosate synthesis, but did not alter macrophage cytokine induction. Our findings s
Elkington P, Shiomi T, Breen R, et al., 2011, MMP-1 drives immunopathology in human tuberculosis and transgenic mice, JOURNAL OF CLINICAL INVESTIGATION, Vol: 121, Pages: 1827-1833, ISSN: 0021-9738
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- Citations: 171
Hickey AJ, Misra A, Robertson BD, 2011, Optimisation of inhaled tuberculosis therapies and implications for host-pathogen interactions, TUBERCULOSIS, Vol: 91, Pages: 64-64, ISSN: 1472-9792
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- Citations: 2
Joyce G, Roberston BD, Williams KJ, 2011, A modified agar pad method for mycobacterial live-cell imaging., BMC Research Notes, Vol: 4
Williams KJ, Boshoff HI, Krishnan N, et al., 2011, The Mycobacterium tuberculosis β-oxidation genes echA5 and fadB3 are dispensable for growth in vitro and in vivo., Tuberculosis, Vol: 91, Pages: 549-555
Nisa S, Blokpoel MCJ, Robertson BD, et al., 2010, Targeting the chromosome partitioning protein ParA in tuberculosis drug discovery, JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY, Vol: 65, Pages: 2347-2358, ISSN: 0305-7453
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- Citations: 28
Krishnan N, Robertson BD, Thwaites G, 2010, The mechanisms and consequences of the extra-pulmonary dissemination of <i>Mycobacterium tuberculosis</i>, TUBERCULOSIS, Vol: 90, Pages: 361-366, ISSN: 1472-9792
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- Citations: 102
Williams KJ, Joyce G, Robertson BD, 2010, Improved mycobacterial tetracycline inducible vectors, PLASMID, Vol: 64, Pages: 69-73, ISSN: 0147-619X
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- Citations: 34
Andreu N, Zelmer A, Fletcher T, et al., 2010, Optimisation of Bioluminescent Reporters for Use with Mycobacteria, PLOS One, Vol: 5, ISSN: 1932-6203
BackgroundMycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.Methodology/Principal FindingsOne of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.Conclusions/SignificanceWhile much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of aut
Robertson BD, 2010, Professor DA Mitchison: 60 years of contributions to the chemotherapy of tuberculosis, TUBERCULOSIS, Vol: 90, Pages: 159-159, ISSN: 1472-9792
Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, et al., 2010, Sensitive Detection of Gene Expression in Mycobacteria under Replicating and Non-Replicating Conditions Using Optimized Far-Red Reporters, PLOS One, Vol: 5, ISSN: 1932-6203
Fluorescent reporter proteins have proven useful for imaging techniques in many organisms. We constructed optimized expression systems for several fluorescent proteins from the far-red region of the spectrum and analyzed their utility in several mycobacterial species. Plasmids expressing variants of the Discosoma Red fluorescent protein (DsRed) from the Mycobacterium bovis hsp60 promoter were unstable; in contrast expression from the Mycobacterium smegmatis rpsA promoter was stable. In Mycobacterium tuberculosis expression of several of the far-red reporters was readily visualised by eye and three reporters (mCherry, tdTomato, and Turbo-635) fluoresced at a high intensity. Strains expressing mCherry showed no fitness defects in vitro or in macrophages. Treatment of cells with antibiotics demonstrated that mCherry could also be used as a reporter for cell death, since fluorescence decreased in the presence of a bactericidal compound, but remained stable in the presence of a bacteriostatic compound. mCherry was functional under hypoxic conditions; using mCherry we demonstrated that the PmtbB is expressed early in hypoxia and progressively down-regulated. mCherry and other far-red fluorescent proteins will have multiple uses in investigating the biology of mycobacteria, particularly under non-replicating, or low cell density conditions, as well as providing a novel means of detecting cell death rapidly.
Berg S, Firdessa R, Habtamu M, et al., 2009, The burden of mycobacterial disease in ethiopian cattle: implications for public health., PLoS One, Vol: 4
BACKGROUND: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a debilitating disease of cattle. Ethiopia has one of the largest cattle populations in the world, with an economy highly dependent on its livestock. Furthermore, Ethiopia has one of the highest incidence rates of human extrapulmonary TB in the world, a clinical presentation that is often associated with transmission of M. bovis from cattle to humans. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a comprehensive investigation of the prevalence of bTB in Ethiopia based on cases identified at slaughterhouses. Out of approximately 32,800 inspected cattle, approximately 4.7% showed suspect tuberculous lesions. Culture of suspect lesions yielded acid-fast bacilli in approximately 11% of cases, with M. bovis accounting for 58 of 171 acid-fast cultures, while 53 isolates were non-tuberculous mycobacteria. Strikingly, M. tuberculosis was isolated from eight cattle, an unusual finding that suggests human to animal transmission. CONCLUSIONS/SIGNIFICANCE: Our analysis has revealed that bTB is widely spread throughout Ethiopia, albeit at a low prevalence, and provides underpinning evidence for public health policy formulation.
Berg S, Firdessa R, Habtamu M, et al., 2009, Correction: The Burden of Mycobacterial Disease in Ethiopian Cattle: Implications for Public Health, PLoS ONE, Vol: 4
Wiles S, Robertson BD, Frankel G, et al., 2009, Bioluminescent Monitoring of In Vivo Colonization and Clearance Dynamics by Light-Emitting Bacteria, BIOLUMINESCENCE: METHODS AND PROTOCOLS, SECOND EDITION, Vol: 574, Pages: 137-153, ISSN: 1064-3745
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- Citations: 17
Marquina-Castillo B, García-García L, Ponce-de-León A, et al., 2008, Virulence, immunopathology and transmissibility of selected strains of Mycobacterium tuberculosis in a murine model., Immunology, Vol: 128, Pages: 123-133
After encounter with Mycobacterium tuberculosis, a series of non-uniform immune responses are triggered that define the course of the infection. Eight M. tuberculosis strains were selected from a prospective population-based study of pulmonary tuberculosis patients (1995–2003) based on relevant clinical/epidemiological patterns and tested in a well-characterized BALB/c mouse model of progressive pulmonary tuberculosis. In addition, a new mouse model of transmissibility consisting of prolonged cohousing (up to 60 days) of infected and naïve animals was tested. Four phenotypes were defined based on strain virulence (mouse survival, lung bacillary load and tissue damage), immunology response (cytokine expression determined by real-time polymerase chain reaction) and transmissibility (lung bacillary loads and cutaneous delayed-type hypersensitivity in naïve animals).We identified four clearly defined strain phenotypes: (1) hypervirulent strain with non-protective immune response and highly transmissible; (2) virulent strain, associated with high expression of proinflammatory cytokines (tumour necrosis factor and interferon) and very low anti-inflammatory cytokine expression (interleukins 4 and 10), which induced accelerated death by immunopathology; (3) strain inducing efficient protective immunity with lower virulence, and (4) strain demonstrating strong and early macrophage activation (innate immunity) with delayed participation of acquired immunity (interferon expression). We were able to correlate virulent and transmissible phenotypes in the mouse model and markers of community transmission such as tuberculin reactivity among contacts, rapid progression to disease and cluster status. However, we were not able to find correlation with the other two phenotypes. Our new transmission model supported the hypothesis that among these strains increased virulence was linked to increased transmission.
Martinez-Gamboa A, Ponce-de-Leon A, Galindo-Fraga A, et al., 2008, Molecular analysis of Mycobacterium tuberculosis strains with an intact pks15/1 gene in a rural community of Mexico, Arch Med Res, Vol: 39, Pages: 809-814
Thanky NR, Young DB, Robertson BD, 2007, Unusual features of the cell cycle in mycobacteria: Polar-restricted growth and the snapping-model of cell division, TUBERCULOSIS, Vol: 87, Pages: 231-236, ISSN: 1472-9792
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- Citations: 96
Stumpf MP, Robertson BD, Duncan K, et al., 2007, Systems biology and its impact on anti-infective drug development., Prog Drug Res, Vol: 64, Pages: 1-20, ISSN: 0071-786X
Systems biology offers the potential for more effective selection of novel targets for anti-infective drugs. In contrast to conventional reductionist biology, a systems approach allows targets to be viewed in a wider context of the entire physiology of the cell, with the potential to identify key susceptible nodes and to predict synergistic effects of blocking multiple pathways. In addition to the holistic perspective provided by systems biology, the emphasis on quantitative analysis is likely to add further rigour to the process of target selection. Systems biology also offers the potential to incorporate different levels of information into the selection process. Consideration of data from microbial population biology may be important in the context of predicting future drug-resistance profiles associated with targeting a particular pathway, for example. This chapter provides an overview of major themes in the developing field of systems biology, summarising the core technologies and the strategies used to translate datasets into useful quantitative models capable of predicting complex biological behaviour.
Stadthagen G, Jackson M, Charles P, et al., 2006, Comparative investigation of the pathogenicity of three <i>Mycobacterium tuberculosis</i> mutants defective in the synthesis of <i>p</i>-hydroxybenzoic acid derivatives, MICROBES AND INFECTION, Vol: 8, Pages: 2245-2253, ISSN: 1286-4579
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- Citations: 20
Wiles S, Hanage WP, Frankel G, et al., 2006, Modelling infectious disease - time to think outside the box?, NATURE REVIEWS MICROBIOLOGY, Vol: 4, Pages: 307-312, ISSN: 1740-1526
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- Citations: 43
Stewart GR, Patel J, Robertson BD, et al., 2005, Mycobacterial mutants with defective control of phagosomal acidification, Plos Pathogens, Vol: 1, Pages: 269-278, ISSN: 1553-7374
The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.
Wiles S, Ferguson K, Stefanidou M, et al., 2005, Alternative luciferase for monitoring bacterial cells under adverse conditions, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 71, Pages: 3427-3432, ISSN: 0099-2240
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- Citations: 43
Murphy HN, Stewart GR, Mischenko VV, et al., 2005, The OtsAB pathway is essential for trehalose biosynthesis in <i>Mycobacterium tuberculosis</i>, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 280, Pages: 14524-14529, ISSN: 0021-9258
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- Citations: 107
Stewart GR, Newton SM, Wilkinson KA, et al., 2005, The stress-responsive chaperone α-crystallin 2 is required for pathogenesis of <i>Mycobacterium tuberculosis</i>, MOLECULAR MICROBIOLOGY, Vol: 55, Pages: 1127-1137, ISSN: 0950-382X
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- Citations: 69
Blokpoel MCJ, Murphy HN, O'Toole R, et al., 2005, Tetracycline-inducible gene regulation in mycobacteria, Nucleic Acids Research, Vol: 33, ISSN: 1362-4962
A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml−1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.
Wiles S, Ferguson K, Robertson B, et al., 2005, Optimisation of conditions for the use of a novel bioluminescent reporter system in <i>Mycobacterium</i> spp., Editors: Tsuji, Matsurnoto, Maeda, Kricka, Stanley, Publisher: WORLD SCIENTIFIC PUBL CO PTE LTD, Pages: 543-546, ISBN: 981-256-118-8
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- Citations: 2
Papatheodorou I, Sergot M, Randall M, et al., 2004, Visualization of microarray results to assist interpretation, TUBERCULOSIS, Vol: 84, Pages: 275-281, ISSN: 1472-9792
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- Citations: 2
Stewart GR, Robertson BD, Young DB, 2004, Analysis of the function of mycobacterial DnaJ proteins by overexpression and microarray profiling, TUBERCULOSIS, Vol: 84, Pages: 180-187, ISSN: 1472-9792
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- Citations: 19
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