Prof Graham Stewart

Professor Graham Stewart


Head of the Department of Microbial Sciences, Professor of Molecular Bacteriology
+44 (0)1483 686423
15 AX 02

Biography

Research interests

Mycobacterium tuberculosis

Tuberculosis (TB) is one of the most important infectious diseases of mankind, claiming 30,000 lives every week. One third of the world's population carry an asymptomatic persistent infection with a 10% risk of progression to active disease. Of the 9 million new cases of tuberculosis every year, more than half a million are caused by strains of Mycobacterium tuberculosis that have acquired multidrug-resistance. BCG is currently the only vaccine used against TB and while it is successful in protecting against disease in children, it is ineffective against adult pulmonary TB. Improvement in diagnosis, drugs and vaccines for tuberculosis will be needed to control this epidemic.

Mycobacterium bovis

Bovine tuberculosis is the most important veterinary health problem in the UK. The projected economic burden to the UK over the next decade is predicted to be £1 billion. Control is likely to require an integrated approach with vaccination of cattle representing a key component. Presently, the M.bovis BCG vaccine represents the most encouraging vaccination option, but it compromises the diagnostic skin test and has a protective efficacy of only ~50-70%.

To form a rational platform upon which to develop novel drugs, vaccines and diagnostic agents we are working to understand the molecular basis of how M. tuberculosis and M.bovis are able to establish infection and generate disease.

 

Research approach

High-throughput functional genomic studies of M.tuberculosis complex bacteria and their host cells by our lab and others have revealed that virulence is controlled by complex multifactorial interactions between thousands of bacterial and host components. From these interaction networks emerge the properties that characterise the pathogenesis of TB. Systems Biology provides a strategy to explore this complexity, integrating experimental analysis and computational tools to develop predictive models that will facilitate effective drug and vaccine development strategies based on a higher level of biological understanding.

We are identifying and studying molecular interaction networks associated with the pathogenesis features below:

 

Mycobacterial inhibition of phagosome maturation

The success of M.tuberculosis/M.bovis as pathogens relies on an ability to grow inside host macrophages. Multiple factors are involved in intracellular survival but a key feature is the ability of M. tuberculosis to arrest the normal process of phagosome maturation, blocking acidification of the intracellular compartment and fusion with lysosomes.

Mycobacterial control of host cell death

Evidence suggests that M.tuberculosis is able to control the fate of host cells such as macrophages,neutrophils and dendritic cells. Early in infection it is able to inhibit apoptosis to preserve its replicative niche but later it has the capacity to induce an inflammatory form of cell death.

Mycobacterial metabolism in host cells/tissues

The features of mycobacterial metabolism during infection are poorly understood. The bacterium exists in many and diverse sites in the host and understanding the variety and nature of metabolic processes utilised in these environments will be essential to novel drug discovery.

The mycobacterial stress response

Pathogenic mycobacteria must endure a variety of other hostile environments during infection. The bacteria counter these harsh conditions with specific and general stress responses that remodel the physiology, biochemistry and structure of the cell. These mechanisms are an essential component of pathogenicity.

Bioarchaeology

In collaboration with Professor Mike Taylor we aim to understand the evolution and macroecology of leprosy and tuberculosis by studying ancient pathogen DNA in archaeological specimens. Characterisation of individual polymorphisms or even reconstruction of whole genome sequences is possible for mycobacterial infections from over 1000 years ago, allowing accurate phylogenetic and evolutionary models to be developed.

Financial support for our research is provided by The Wellcome Trust, the BBSRC and the Rosetrees Trust.

Research collaborations

 

Main internal collaborators:

Johnjoe McFadden

Andrzej Kierzek

Nicolas Locker

Colin Smith

Teaching

 

Graduate

BMS1026 Microbiology: An Introduction to the Microbial World

BMS1031 Introduction to Molecular Biology and Genetics

BMS2037 Cellular Microbiology and Virology

BMS3079 Human Microbial Diseases

 

Postgraduate

MSc Medical Microbiology

MMIM018 Microbial Genetics and Molecular Biology

MMIM026 Research Methods 1

MMIM024 Pathogenesis of infectious disease

Departmental duties

 

Infectious Diseases Research Group Leader

Chair Board of Examinations MSc Medical Microbiology Programmes

Academic Lead for Bioimaging and Flow Cytometry Facility link

My publications

Publications

Wu Huihai, von Kamp A, Leoncikas Vytautas, Mori W, Sahin N, Gevorgyan A, Linley C, Grabowski M, Mannan AA, Stoy Nicholas, Stewart Graham, Ward LT, Lewis David, Sroka J, Matsuno H, Klamt S, Westerhoff HV, McFadden Johnjoe, Plant NJ, Kierzek Andrzej (2016) MUFINS: Multi-Formalism Interaction Network Simulator, NPJ Systems Biology and Applications 2 16032 Nature Publishing Group
Systems Biology has established numerous approaches for mechanistic modelling of molecular networks in the cell and a legacy of models. The current frontier is the integration of models expressed in different formalisms to address the multi-scale biological system organisation challenge. We present MUFINS software, implementing a unique set of approaches for multiformalism simulation of interaction networks. We extend the constraint-based modelling (CBM) framework by incorporation of linear inhibition constraints, enabling for the first time linear modelling of networks simultaneously describing gene regulation, signalling and whole-cell metabolism at steady state. We present a use case where a logical hypergraph model of a regulatory network is expressed by linear constraints and integrated with a Genome Scale Metabolic Network (GSMN) of mouse macrophage. We experimentally validate predictions, demonstrating application of our software in an iterative cycle of hypothesis generation, validation and model refinement. MUFINS incorporates an extended version of our Quasi Steady State Petri Net approach to integrate dynamic models with CBM, which we demonstrate through a dynamic model of cortisol signalling integrated with the human Recon2 GSMN and a model of nutrient dynamics in physiological compartments. Finally, we implement a number of methods for deriving metabolic states from ~omics data, including our new variant of the iMAT congruency approach. We compare our approach with iMAT through analysis of 262 individual tumour transcriptomes, recovering features of metabolic reprogramming in cancer. The software provides graphics user interface with network visualisation, which facilitates use by researchers who are not experienced in coding and mathematical modelling environments.
Bradley JE, Elson L, Tree TI, Stewart G, Guderian R, Calvopiña M, Paredes W, Araujo E, Nutman TB (1995) Resistance to Onchocerca volvulus: differential cellular and humoral responses to a recombinant antigen, OvMBP20/11., J Infect Dis 172 (3) pp. 831-837
Persons putatively immune (PI) to Onchocerca volvulus (Ov) infection were identified in Ecuador on the basis of epidemiologic, clinical, and parasitologic findings. Immune responses of PI subjects to a recombinant onchocercal protein, OvMBP20/11, were determined and compared with those of a comparable infected (INF) group from the same Ov-endemic area. PI subjects had significantly less antibody reactivity to this molecule; however, not all INF subjects had an antibody response. IgG1 and IgG4 were the predominant IgG subclasses induced to this molecule, and the amount of IgG1 produced was the only significant difference between the PI and INF groups. In contrast to the antibody responses, proliferative responses to OvMBP20/11 were significantly higher in PI than in INF subjects. Cytokine analysis of peripheral blood mononuclear cell culture supernatants revealed that INF subjects produced significantly more interleukin-10 in response to OvMBP20/11 than did PI subjects. This antigen induced few other cytokines, and there were no differences between study groups.
Stewart GR, Snewin VA, Walzl G, Hussell T, Tormay P, O'Gaora P, Goyal M, Betts J, Brown IN, Young DB (2001) Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection., Nat Med 7 (6) pp. 732-737
Elevated expression of heat-shock proteins (HSPs) can benefit a microbial pathogen struggling to penetrate host defenses during infection, but at the same time might provide a crucial signal alerting the host immune system to its presence. To determine which of these effects predominate, we constructed a mutant strain of Mycobacterium tuberculosis that constitutively overexpresses Hsp70 proteins. Although the mutant was fully virulent in the initial stage of infection, it was significantly impaired in its ability to persist during the subsequent chronic phase. Induction of microbial genes encoding HSPs might provide a novel strategy to boost the immune response of individuals with latent tuberculosis infection.
BACKGROUND: Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host's immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive. RESULTS: Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs. CONCLUSION: We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.
Stark A-K, Bermudez-Fajardo A, El Kadri R, Stewart G, Oviedo-Orta E (2010) Study of the immunomodulatory properties of the major outer membrane protein (MOMP) of Chlamydophila pneumoniae in the context of atherosclerosis, IMMUNOLOGY 131 pp. 30-30 WILEY-BLACKWELL PUBLISHING, INC
Stark A-K, Bermudez-Fajardo A, El Kadri R, Stewart G, Oviedo-Orta E (2010) Study of the immunomodulatory properties of the Major Outer Membrane Protein (MOMP) of Chlamydophila pneumoniae in the context of atherosclerosis, IMMUNOLOGY 131 pp. 110-111 WILEY-BLACKWELL PUBLISHING, INC
Dussurget O, Stewart G, Neyrolles O, Pescher P, Young D, Marchal G (2001) Role of Mycobacterium tuberculosis copper-zinc superoxide dismutase, INFECTION AND IMMUNITY 69 (1) pp. 529-533 AMER SOC MICROBIOLOGY
Mendum TA, Schuenemann VJ, Roffey S, Taylor GM, Wu H, Singh P, Tucker K, Hinds J, Cole ST, Kierzek AM, Nieselt K, Krause J, Stewart GR (2014) Mycobacterium leprae genomes from a British medieval leprosy hospital: Towards understanding an ancient epidemic, BMC Genomics 15 (1)
Background: Leprosy has afflicted humankind throughout history leaving evidence in both early texts and the archaeological record. In Britain, leprosy was widespread throughout the Middle Ages until its gradual and unexplained decline between the 14th and 16th centuries. The nature of this ancient endemic leprosy and its relationship to modern strains is only partly understood. Modern leprosy strains are currently divided into 5 phylogenetic groups, types 0 to 4, each with strong geographical links. Until recently, European strains, both ancient and modern, were thought to be exclusively type 3 strains. However, evidence for type 2 strains, a group normally associated with Central Asia and the Middle East, has recently been found in archaeological samples in Scandinavia and from two skeletons from the medieval leprosy hospital (or leprosarium) of St Mary Magdalen, near Winchester, England.Results: Here we report the genotypic analysis and whole genome sequencing of two further ancient M. leprae genomes extracted from the remains of two individuals, Sk14 and Sk27, that were excavated from 10th-12th century burials at the leprosarium of St Mary Magdalen. DNA was extracted from the surfaces of bones showing osteological signs of leprosy. Known M. leprae polymorphisms were PCR amplified and Sanger sequenced, while draft genomes were generated by enriching for M. leprae DNA, and Illumina sequencing. SNP-typing and phylogenetic analysis of the draft genomes placed both of these ancient strains in the conserved type 2 group, with very few novel SNPs compared to other ancient or modern strains.Conclusions: The genomes of the two newly sequenced M. leprae strains group firmly with other type 2F strains. Moreover, the M. leprae strain most closely related to one of the strains, Sk14, in the worldwide phylogeny is a contemporaneous ancient St Magdalen skeleton, vividly illustrating the epidemic and clonal nature of leprosy at this site. The prevalence of these type 2 strains indicates that type 2F strains, in contrast to later European and associated North American type 3 isolates, may have been the co-dominant or even the predominant genotype at this location during the 11th century. © 2014 Mendum et al.; licensee BioMed Central Ltd.
Stewart GR, Robertson BD, Young DB (2004) Analysis of the function of mycobacterial DnaJ proteins by overexpression and microarray profiling., Tuberculosis (Edinb) 84 (3-4) pp. 180-187
Regulation of expression of the Hsp70/DnaK chaperone plays an important role during infection with Mycobacterium tuberculosis. We have examined the effect of manipulating the level of expression of DnaJ, one of the components of the chaperone apparatus. Overexpression of DnaJ1 resulted in elevated transcription of both the hsp70/dnaK and hsp60/groE chaperone genes, consistent with an increase in the cellular content of nascent and unfolded peptide substrates. There was also an increase in transcription of genes flanking the origin of chromosomal replication, suggesting an important role for DnaJ1 in controlling interaction of the Hsp70 chaperone with the DnaA protein. Overexpression of DnaJ2 had no detectable effect on transcription of other genes. Overexpression in combination with microarray profiling provides a complementary approach to gene deletion for exploring the function of essential genes in M. tuberculosis.
Stewart GR, Patel J, Robertson BD, Rae A, Young DB (2005) Mycobacterial mutants with defective control of phagosomal acidification, PLOS PATHOGENS 1 (3) ARTN e33 pp. 269-278 PUBLIC LIBRARY SCIENCE
Stewart GR, Robertson BD, Young DB (2003) Tuberculosis: a problem with persistence., Nat Rev Microbiol 1 (2) pp. 97-105
Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year. The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease. The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M. tuberculosis dates back to the first half of the last century. Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.
Stewart GR, Zhu Y, Parredes W, Tree TI, Guderian R, Bradley JE (1997) The novel cuticular collagen Ovcol-1 of Onchocerca volvulus is preferentially recognized by immunoglobulin G3 from putatively immune individuals., Infect Immun 65 (1) pp. 164-170
The cDNA sequence encoding an Onchocerca volvulus collagen, Ovcol-1, has been isolated and the corresponding native antigen has been identified. The cDNA encodes an open reading frame of 96 amino acid residues containing an uninterrupted 66-residue Gly-X-Y repeat triple-helical (TH) domain (where X and Y may be any amino acids) flanked by a 26-residue amino non-TH domain and a 4-residue carboxyl non-TH domain. The size (9.7 kDa) and structure of the deduced molecule are unique among previously identified collagen chains. This novel collagen type has been designated "mini-chain collagen." Native Ovcol-1 is aqueous soluble and resolves by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 14.2 kDa under reducing conditions. Immunoelectron microscopy of adult female O. volvulus localized Ovcol-1 to the cuticles of both the adult worm and uterine microfilaria. A group of individuals from an area in Ecuador where O. volvulus is hyperendemic have been classified as putatively immune (PI) to O. volvulus infection. Analysis of the humoral immune responses to Ovcol-1 demonstrated that immunoglobulin G3 (IgG3) of PI individuals preferentially recognized this antigen in comparison to IgG3 of infected individuals.
Humphreys IR, Stewart GR, Turner DJ, Patel J, Karamanou D, Snelgrove RJ, Young DB (2006) A role for dendritic cells in the dissemination of mycobacterial infection., Microbes Infect 8 (5) pp. 1339-1346
The ability of mycobacteria to disseminate from the initial site of infection has an important role in immune priming and in the seeding of disease in multiple organs. To study this phenomenon, we used flow cytometry to analyse the distribution of green fluorescent protein-labelled BCG amongst different populations of antigen-presenting cells in the lungs of mice following intranasal infection, and monitored appearance of live bacteria in the draining mediastinal lymph nodes. BCG predominantly infected alveolar macrophages (CD11c(+)/CD11b(-)) and dendritic cells (CD11c(+)/CD11b(+)) in the lungs. The bacteria that disseminated to the lymph node were found in dendritic cells. The results are consistent with a model in which mycobacterial dissemination from the lung is initiated by the migration of infected dendritic cells to the draining lymph nodes.
El Kadri R, Bermudez-Fajardo A, Puolakkainen M, Stewart G, Oviedo-Orta E (2010) Anti-inflammatory and atheroprotective effects of Chlamydia pneumoniae recombinant MOMP, ATHEROSCLEROSIS 213 (1) pp. E6-E6 ELSEVIER IRELAND LTD
Stewart GR, Ehrt S, Riley LW, Dale JW, McFadden J (2000) Deletion of the putative antioxidant noxR1 does not alter the virulence of Mycobacterium tuberculosis H37Rv, TUBERCLE AND LUNG DISEASE 80 (4-5) pp. 237-242 CHURCHILL LIVINGSTONE
Snelgrove RJ, Cornere MM, Edwards L, Dagg B, Keeble J, Rodgers A, Lyonga DE, Stewart GR, Young DB, Walker B, Hussell T (2012) OX40 ligand fusion protein delivered simultaneously with the BCG vaccine provides superior protection against murine Mycobacterium tuberculosis infection., Journal of Infectious Diseases 205 (6) pp. 975-983 OXFORD UNIV PRESS INC
Mycobacterium tuberculosis infection claims approximately 2 million lives per year, and improved efficacy of the BCG vaccine remains a World Health Organization priority. Successful vaccination against M. tuberculosis requires the induction and maintenance of T cells. Targeting molecules that promote T-cell survival may therefore provide an alternative strategy to classic adjuvants. We show that the interaction between T-cell-expressed OX40 and OX40L on antigen-presenting cells is critical for effective immunity to BCG. However, because OX40L is lost rapidly from antigen-presenting cells following BCG vaccination, maintenance of OX40-expressing vaccine-activated T cells may not be optimal. Delivering an OX40L:Ig fusion protein simultaneously with BCG provided superior immunity to intravenous and aerosol M. tuberculosis challenge even 6 months after vaccination, an effect that depends on natural killer 1.1(+) cells. Attenuated vaccines may therefore lack sufficient innate stimulation to maintain vaccine-specific T cells, which can be replaced by reagents binding inducible T-cell costimulators.
Young DB, Stewart GR (2002) Tuberculosis vaccines., Br Med Bull 62 pp. 73-86
The increasing incidence of disease associated with HIV infection highlights the crucial role of the immune response in susceptibility to tuberculosis and has stimulated renewed efforts to develop improved vaccines. Vaccine targets include prevention of infection in naive individuals, prevention of re-activation in individuals harbouring latent infection, and prevention of relapse by immunotherapy in tuberculosis patients. Advances in mycobacterial molecular genetics have facilitated development of a range of live attenuated and subunit vaccine candidates that have been screened in experimental models of infection. Evaluation of the immunogenicity of selected candidate vaccines in clinical trials should be combined with a continuation of fundamental research on the immune response to mycobacterial infection and persistence.
El Kadri R, Bermudez-Fajardo A, Puolakkainen M, Stewart G, Oviedo-Orta E (2010) Effect of immunisation with C. pneumoniae rMOMP on atherosclerosis development, ATHEROSCLEROSIS 213 (1) pp. E13-E13 ELSEVIER IRELAND LTD
Butler RE, Cihlarova V, Stewart GR (2010) Effective generation of reactive oxygen species in the mycobacterial phagosome requires K plus efflux from the bacterium, CELLULAR MICROBIOLOGY 12 (8) pp. 1186-1193 WILEY-BLACKWELL
Summary: Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non-pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCG” kef), deficient in a Kef-type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild-type BCG, and the production of ROS by macrophages infected with BCG” kef is substantially decreased compared with those infected with wild-type BCG. Our results suggest that increased intracellular survival of BCG” kef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS. © 2010 Blackwell Publishing Ltd.
Pitarque S, Herrmann JL, Duteyrat JL, Jackson M, Stewart GR, Lecointe F, Payre B, Schwartz O, Young DB, Marchal G, Lagrange PH, Puzo G, Gicquel B, Nigou J, Neyrolles O (2005) Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity, BIOCHEMICAL JOURNAL 392 pp. 615-624 PORTLAND PRESS LTD
Stewart GR, Boussinesq M, Coulson T, Elson L, Nutman T, Bradley JE (1999) Onchocerciasis modulates the immune response to mycobacterial antigens., Clin Exp Immunol 117 (3) pp. 517-523
Chronic helminth infection induces a type-2 cellular immune response. In contrast to this, mycobacterial infections commonly induce a type-1 immune response which is considered protective. Type-2 responses and diminished type-1 responses to mycobacteria have been previously correlated with active infection states such as pulmonary tuberculosis and lepromatous leprosy. The present study examines the immune responses of children exposed to both the helminth parasite Onchocerca volvulus and the mycobacterial infections, Mycobacterium tuberculosis and M. leprae. Proliferation of peripheral blood mononuclear cells (PBMC) and production of IL-4 in response to both helminth and mycobacterial antigen (PPD) decreased dramatically with increasing microfilarial (MF) density. Although interferon-gamma (IFN-gamma) production strongly correlated with cellular proliferation, it was surprisingly not related to MF density for either antigen. IL-4 production in response to helminth antigen and PPD increased with ascending children's age. IFN-gamma and cellular proliferation to PPD were not related to age, but in response to helminth antigen were significantly higher in children of age 9-12 years than children of either the younger age group (5-8 years) or the older group (13-16 years). Thus, there was a MF density-related down-regulation of cellular responsiveness and age-related skewing toward type 2 which was paralleled in response to both the helminth antigen and PPD. This parasite-induced immunomodulation of the response to mycobacteria correlates with a previous report of doubled incidence of lepromatous leprosy in onchocerciasis hyperendemic regions. Moreover, this demonstration that helminth infection in humans can modulate the immune response to a concurrent infection or immunological challenge is of critical importance to future vaccination strategies.
Yeremeev VV, Stewart GR, Neyrolles O, Skrabal K, Avdienko VG, Apt AS, Young DB (2000) Deletion of the 19kDa antigen does not alter the protective efficacy of BCG, TUBERCLE AND LUNG DISEASE 80 (6) pp. 243-247 CHURCHILL LIVINGSTONE
Butler RE, Brodin P, Jang J, Jang MS, Robertson BD, Gicquel B, Stewart GR (2012) The balance of apoptotic and necrotic cell death in Mycobacterium tuberculosis infected macrophages is not dependent on bacterial virulence., PLoS One 7 (10) Public Library of Science
An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.
Ciaramella A, Cavone A, Santucci MB, Garg SK, Sanarico N, Bocchino M, Galati D, Martino A, Auricchio G, D'Orazio M, Stewart GR, Neyrolles O, Young DB, Colizzi V, Fraziano M (2004) Induction of apoptosis and release of interleukin-1 beta by cell wall-associated 19-kDa lipoprotein during the course of mycobacterial infection, JOURNAL OF INFECTIOUS DISEASES 190 (6) pp. 1167-1176 UNIV CHICAGO PRESS
Blokpoel MC, Murphy HN, O'Toole R, Wiles S, Runn ES, Stewart GR, Young DB, Robertson BD (2005) Tetracycline-inducible gene regulation in mycobacteria., Nucleic Acids Res 33 (2)
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.
El-Kadri R, Bermudez-Fajardo A, Stewart G, Puolakkainen M, Oviedo-Orta E (2010) Anti-inflammatory and atheroprotectiveeffects of Chlamyiphia pneumoniaerecombinant MOMP, IMMUNOLOGY 131 pp. 131-131 WILEY-BLACKWELL PUBLISHING, INC
Stewart GR, Patel J, Robertson BD, Rae A, Young DB (2005) Mycobacterial mutants with defective control of phagosomal acidification, PLoS Pathogens 1 (3) pp. 0269-0278
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 virulenceassociated 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. © 2005 Stewart et al.
Mendum TA, Schuenemann VJ, Roffey S, Taylor GM, Wu H, Singh P, Tucker K, Hinds J, Cole ST, Kierzek AM, Nieselt K, Krause J, Stewart GR (2014) Mycobacterium leprae genomes from a British medieval leprosy hospital: towards understanding an ancient epidemic., BMC Genomics 15
BACKGROUND: Leprosy has afflicted humankind throughout history leaving evidence in both early texts and the archaeological record. In Britain, leprosy was widespread throughout the Middle Ages until its gradual and unexplained decline between the 14th and 16th centuries. The nature of this ancient endemic leprosy and its relationship to modern strains is only partly understood. Modern leprosy strains are currently divided into 5 phylogenetic groups, types 0 to 4, each with strong geographical links. Until recently, European strains, both ancient and modern, were thought to be exclusively type 3 strains. However, evidence for type 2 strains, a group normally associated with Central Asia and the Middle East, has recently been found in archaeological samples in Scandinavia and from two skeletons from the medieval leprosy hospital (or leprosarium) of St Mary Magdalen, near Winchester, England. RESULTS: Here we report the genotypic analysis and whole genome sequencing of two further ancient M. leprae genomes extracted from the remains of two individuals, Sk14 and Sk27, that were excavated from 10th-12th century burials at the leprosarium of St Mary Magdalen. DNA was extracted from the surfaces of bones showing osteological signs of leprosy. Known M. leprae polymorphisms were PCR amplified and Sanger sequenced, while draft genomes were generated by enriching for M. leprae DNA, and Illumina sequencing. SNP-typing and phylogenetic analysis of the draft genomes placed both of these ancient strains in the conserved type 2 group, with very few novel SNPs compared to other ancient or modern strains. CONCLUSIONS: The genomes of the two newly sequenced M. leprae strains group firmly with other type 2F strains. Moreover, the M. leprae strain most closely related to one of the strains, Sk14, in the worldwide phylogeny is a contemporaneous ancient St Magdalen skeleton, vividly illustrating the epidemic and clonal nature of leprosy at this site. The prevalence of these type 2 strains indicates that type 2F strains, in contrast to later European and associated North American type 3 isolates, may have been the co-dominant or even the predominant genotype at this location during the 11th century.
Beste DJV, Espasa M, Bonde B, Kierzek AM, Stewart GR, McFadden J (2009) The Genetic Requirements for Fast and Slow Growth in Mycobacteria, PLOS ONE 4 (4) ARTN e5349 PUBLIC LIBRARY SCIENCE
Bradley JE, Atogho BM, Elson L, Stewart GR, Boussinesq M (1998) A cocktail of recombinant Onchocerca volvulus antigens for serologic diagnosis with the potential to predict the endemicity of onchocerciasis infection., Am J Trop Med Hyg 59 (6) pp. 877-882
We report here the evaluation of the potential of a serologic test to determine the endemicity of onchocercal infection in hyper, meso, and hypoendemic communities by the detection of antibodies to a cocktail of recombinant antigens. Parasitologic parameters of infection prevalence and intensity were compared with serologic results. Infection prevalence by serology was consistently but not significantly higher than that defined by parasitology. Differences between the communities defined by microfilarial load (CMFL) and a measurement of Onchocerca volvulus-specific antibody levels (serologic index [SI]) were similar. When stratified by age, differences were more significant in the younger age groups. If a sentinel population of 5-15-year-old individuals was used to compare communities, all could be equally ranked by serologic and parasitologic parameters. The SI of the sentinel population gave a better distinction between each community than the SI of the whole and would be sufficiently sensitive to measure the changes in endemicity that would be required for onchocerciasis control programs.
Brodin P, Poquet Y, Levillain F, Peguillet I, Larrouy-Maumus G, Gilleron M, Ewann F, Christophe T, Fenistein D, Jang J, Jang M-S, Park S-J, Rauzier J, Carralot J-P, Shrimpton R, Genovesio A, Gonzalo-Asensio JA, Puzo G, Martin C, Brosch R, Stewart GR, Gicquel B, Neyrolles O (2010) High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling, PLOS PATHOGENS 6 (9) ARTN e1001100 PUBLIC LIBRARY SCIENCE
Taylor GM, Tucker K, Butler R, Pike AW, Lewis J, Roffey S, Marter P, Lee OY, Wu HH, Minnikin DE, Besra GS, Singh P, Cole ST, Stewart GR (2013) Detection and strain typing of ancient Mycobacterium leprae from a medieval leprosy hospital., PLoS One 8 (4)
Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.
STEWART GR, PERRY RN, WRIGHT DJ (1993) STUDIES ON THE AMPHID SPECIFIC GLYCOPROTEIN GP32 IN DIFFERENT LIFE-CYCLE STAGES OF MELOIDOGYNE SPECIES, PARASITOLOGY 107 pp. 573-578 CAMBRIDGE UNIV PRESS
STEWART GR, PERRY RN, ALEXANDER J, WRIGHT DJ (1993) A GLYCOPROTEIN SPECIFIC TO THE AMPHIDS OF MELOIDOGYNE SPECIES, PARASITOLOGY 106 pp. 405-412 CAMBRIDGE UNIV PRESS
Stewart GR, Elson L, Araujo E, Guderian R, Nutman TB, Bradley JE (1995) Isotype-specific characterization of antibody responses to Onchocerca volvulus in putatively immune individuals., Parasite Immunol 17 (7) pp. 371-380
Isotype/subclass-specific antibody responses to adult Onchocerca volvulus extract (OvAg) were assessed by both ELISA and immunoblotting for a group of putatively immune individuals (PIs, n = 29) from a hyperendemic area in Ecuador and for a group of infected individuals (INFs, n = 470) from the same regions. As a group, the PIs have been previously shown to possess lower levels of OvAg specific IgG1, IgG2, IgG3 and IgG4 than INF's but semi-quantitative analysis revealed that the relative proportions of these subclasses differs between the two groups. The IgG of the PI group contained a higher proportion of IgG3 and a lower proportion of IgG4 than the INF group. The frequency distribution of IgG3 responses was similar for the PI and INF groups. The frequency distributions for IgG1, IgG4 and IgE were significantly different between the PI and INF groups. A subgroup of the PIs were identified from frequency distributions and multivariate plots of individual isotype responses as having antibody responses (mainly IgG4) possibly indicative of cryptic infection. High IgE responses were exclusive to INF individuals, and a rare response type of high IgG3 with negligible levels of other isotypes/subclasses was seen only in the PI group. However, the majority of the PIs had negligible responses for all antibody classes. Immunoblots demonstrated no obvious differences in qualitative recognition between the PIs and INFs.
Inskip SA, Taylor GM, Zakrzewski SR, Mays SA, Pike AW, Llewellyn G, Williams CM, Lee OY, Wu HH, Minnikin DE, Besra GS, Stewart GR (2015) Osteological, biomolecular and geochemical examination of an early anglo-saxon case of lepromatous leprosy., PLoS One 10 (5)
We have examined a 5th to 6th century inhumation from Great Chesterford, Essex, UK. The incomplete remains are those of a young male, aged around 21-35 years at death. The remains show osteological evidence of lepromatous leprosy (LL) and this was confirmed by lipid biomarker analysis and ancient DNA (aDNA) analysis, which provided evidence for both multi-copy and single copy loci from the Mycobacterium leprae genome. Genotyping showed the strain belonged to the 3I lineage, but the Great Chesterford isolate appeared to be ancestral to 3I strains found in later medieval cases in southern Britain and also continental Europe. While a number of contemporaneous cases exist, at present, this case of leprosy is the earliest radiocarbon dated case in Britain confirmed by both aDNA and lipid biomarkers. Importantly, Strontium and Oxygen isotope analysis suggest that the individual is likely to have originated from outside Britain. This potentially sheds light on the origins of the strain in Britain and its subsequent spread to other parts of the world, including the Americas where the 3I lineage of M. leprae is still found in some southern states of America.
Mokhtar H, Pedrera M, Frossard J-P, Biffar L, Hammer SE, Kvisgaard LK, Larsen LE, Stewart GR, Somavarapu S, Steinbach F, Graham SP (2016) The Non-structural Protein 5 and Matrix Protein Are Antigenic Targets of T Cell Immunity to Genotype 1 Porcine Reproductive and Respiratory Syndrome Viruses, FRONTIERS IN IMMUNOLOGY 7 ARTN 40 FRONTIERS MEDIA SA
Wilkinson KA, Stewart GR, Newton SM, Vordermeier HM, Wain JR, Murphy HN, Horner K, Young DB, Wilkinson RJ (2005) Infection biology of a novel alpha-crystallin of Mycobacterium tuberculosis: Acr2, JOURNAL OF IMMUNOLOGY 174 (7) pp. 4237-4243 AMER ASSOC IMMUNOLOGISTS
Newton SM, Smith RJ, Wilkinson KA, Nicol MP, Garton NJ, Staples KJ, Stewart GR, Wain JR, Martineau AR, Fandrich S, Smallie T, Foxwell B, Al-Obaidi A, Shafi J, Rajakumar K, Kampmann B, Andrew PW, Ziegler-Heitbrock L, Barer MR, Wilkinson RJ (2006) A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion., Proc Natl Acad Sci U S A 103 (42) pp. 15594-15598
Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.
Stewart GR, Perry RN, Wright DJ (2001) Occurrence of dopamine in Panagrellus redivivus and Meloidogyne incognita, NEMATOLOGY 3 pp. 843-848 BRILL ACADEMIC PUBLISHERS
Murphy HN, Stewart GR, Mischenko VV, Apt AS, Harris R, McAlister MS, Driscoll PC, Young DB, Robertson BD (2005) The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis., J Biol Chem 280 (15) pp. 14524-14529
The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from alpha-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.
Stewart GR, Young DB (2004) Heat-shock proteins and the host-pathogen interaction during bacterial infection., Curr Opin Immunol 16 (4) pp. 506-510
Heat-shock proteins (HSPs) are expressed at high levels by bacterial pathogens during adaptation to intracellular survival. Both host and pathogen heat-shock proteins contribute to immunity by receptor-mediated activation of the innate immune response and by participation in the presentation of antigens for the adaptive immune response. Manipulation of these interactions presents a potential route to improved control of infection by vaccination or immunotherapy.
Elkadri RAS, Grobler A-K, Bermudez-Fajardo A, Puolakkainen M, Stewart G, Oviedo-Orta E (2009) Effects of recombinant vectors expressing Chlamydia pneumoniae's major outer membrane protein (MOMP) on humoral and T cell-mediated immunity., JOURNAL OF IMMUNOLOGY 182 AMER ASSOC IMMUNOLOGISTS
Schuenemann VJ, Bos KI, Krause J, Singh P, Benjak A, Busso P, Cole ST, Mendum TA, Wu H, Stewart GR, Taylor GM, Krause-Kyora B, Nebel A, Jäger G, Herbig A, Nieselt K, Economou C, Boldsen JL, Kjellström A, Bauer P, Lee OY-C, Wu HHT, Minnikin DE, Besra GS, Tucker K, Roffey S, Sow SO (2013) Genome-wide comparison of medieval and modern Mycobacterium leprae, Science 341 (6142) pp. 179-183
Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.
Stewart GR, Wernisch L, Stabler R, Mangan JA, Hinds J, Laing KG, Butcher PD, Young DB (2002) The heat shock response of Mycobacterium tuberculosis: linking gene expression, immunology and pathogenesis., Comp Funct Genomics 3 (4) pp. 348-351
The regulation of heat shock protein (HSP) expression is critically important to pathogens such as Mycobacterium tuberculosis and dysregulation of the heat shock response results in increased immune recognition of the bacterium and reduced survival during chronic infection. In this study we use a whole genome spotted microarray to characterize the heat shock response of M. tuberculosis. We also begin a dissection of this important stress response by generating deletion mutants that lack specific transcriptional regulators and examining their transcriptional profiles under different stresses. Understanding the stimuli and mechanisms that govern heat shock in mycobacteria will allow us to relate observed in vivo expression patterns of HSPs to particular stresses and physiological conditions. The mechanisms controlling HSP expression also make attractive drug targets as part of a strategy designed to enhance immune recognition of the bacterium.
Papatheodorou I, Sergot M, Randall M, Stewart GR, Robertson BD (2004) Visualization of microarray results to assist interpretation., Tuberculosis (Edinb) 84 (3-4) pp. 275-281
Whole genome microarrays allow assessment of the profile of genes expressed under particular experimental conditions, including external stimuli such as pH or temperature, and internal changes brought about by deleting or over-expressing a gene. Such experiments produce large data sets, for which sophisticated analysis software is available. What is lacking are tools for analysing data sets from different experiments, in order to test and generate hypotheses about the links between regulatory networks. We describe here a method for presenting results from different experiments as a directed graph constructed using an automated graph drawing program xneato, enhanced by a logic program designed to cluster data and aid in the generation of hypotheses about possible gene interactions. A web-based front-end to the system has been constructed to explore and manipulate the graphical displays produced. Results of microarray experiments on Mycobacterium tuberculosis were used to develop and evaluate the visualization tool and initiate the development of an inference system for gene interactions based on such data. The GeneGraph project can be accessed at: zebrafish.doc.ic.ac.uk
Estorninho M, Smith H, Thole J, Harders-Westerveen J, Kierzek A, Butler RE, Neyrolles O, Stewart GR (2010) ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage, Microbiology 156 (11) pp. 3445-3455 Society for General Microbiology
Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1?h post-phagocytosis.
Stewart GR, Newton SM, Wilkinson KA, Humphreys IR, Murphy HN, Robertson BD, Wilkinson RJ, Young DB (2005) The stress-responsive chaperone alpha-crystallin 2 is required for pathogenesis of Mycobacterium tuberculosis, MOLECULAR MICROBIOLOGY 55 (4) pp. 1127-1137 BLACKWELL PUBLISHING LTD
Walters AA, Somavarapu S, Riitho V, Stewart GR, Charleston B, Steinbach F, Graham SP (2015) Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody, VACCINE 33 (48) pp. 6588-6595 ELSEVIER SCI LTD
Schuenemann VJ, Singh P, Mendum TA, Krause-Kyora B, Jäger G, Bos KI, Herbig A, Economou C, Benjak A, Busso P, Nebel A, Boldsen JL, Kjellström A, Wu H, Stewart GR, Taylor GM, Bauer P, Lee OY, Wu HH, Minnikin DE, Besra GS, Tucker K, Roffey S, Sow SO, Cole ST, Nieselt K, Krause J (2013) Genome-wide comparison of medieval and modern Mycobacterium leprae., Science 341 (6142) pp. 179-183
Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.
Stewart GR, Wernisch L, Stabler R, Mangan JA, Hinds J, Laing KG, Young DB, Butcher PD (2002) Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays., Microbiology 148 (Pt 10) pp. 3129-3138
Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass alpha-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heat-shock response and is related to a major antigen induced in response to anaerobic stress.
Francis RJ, Butler RE, Stewart GR (2014) Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx, necrosis and neutrophil extracellular trap formation., Cell Death Dis 5
Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca(2+) overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca(2+) activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca(2+), stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.
Henao-Tamayo M, Junqueira-Kipnis AP, Ordway D, Gonzales-Juaffero M, Stewart GR, Young DB, Wilkinson RJ, Basaraba RJ, Orme IM (2007) A mutant of Mycobacterium tuberculosis lacking the 19-kDa lipoprotein Rv3763 is highly attenuated in vivo but retains potent vaccinogenic properties, VACCINE 25 (41) pp. 7153-7159 ELSEVIER SCI LTD
STEWART GR, PERRY RN, WRIGHT DJ (1994) IMMUNOCYTOCHEMICAL STUDIES ON THE OCCURRENCE OF GAMMA-AMINOBUTYRIC-ACID IN THE NERVOUS-SYSTEM OF THE NEMATODES PANAGRELLUS-REDIVIVUS, MELOIDOGYNE-INCOGNITA AND GLOBODERA-ROSTOCHIENSIS, FUNDAMENTAL AND APPLIED NEMATOLOGY 17 (5) pp. 433-439 GAUTHIER-VILLARS
Minnikin DE, Taylor GM, Stewart GR, Mendum TA, Roffey S, Tucker K, Lee OY-C, Wu HHT, Besra GS, Cole ST (2014) Defining the origins and spread of leprosy using molecular biomarkers, AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 153 pp. 186-187 WILEY-BLACKWELL
Wilkinson KA, Newton SM, Stewart GR, Martineau AR, Patel J, Sullivan SM, Herrmann J-L, Neyrolles O, Young DB, Wilkinson RJ (2009) Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis, BMC MICROBIOLOGY 9 ARTN 93 BIOMED CENTRAL LTD
Stark A, Bermudez-Fajardo A, El Kadri R, Stewart G, Oviedo-Orta E (2010) Study of the immunomodulatory properties of the major outer membrane protein (MOMP) of Chlamydophila pneumoniae in the context of atherosclerosis, ATHEROSCLEROSIS 213 (1) pp. E10-E11 ELSEVIER IRELAND LTD
Taylor GM, Stewart GR, Cooke M, Chaplin S, Ladva S, Kirkup J, Palmer S, Young DB (2003) Koch's Bacillus - a look at the first isolate of Mycobacterium tuberculosis from a modern perspective, MICROBIOLOGY-SGM 149 pp. 3213-3220 SOC GENERAL MICROBIOLOGY
Beste DJV, Hooper T, Stewart G, Bonde B, Avignone-Rossa C, Bushell M, Wheeler P, Klamt S, Kierzek AM, McFadden J (2007) GSMN-TB: a web-based genome scale network model of Mycobacterium tuberculosis metabolism, GENOME BIOLOGY 8 (5) ARTN r89 BIOMED CENTRAL LTD
Bhatt A, Stewart GR, Kieser T (2002) Transposition of Tn4560 of Streptomyces fradiae in Mycobacterium smegmatis, FEMS MICROBIOLOGY LETTERS 206 (2) pp. 241-246 ELSEVIER SCIENCE BV
Stewart GR, Wilkinson KA, Newton SM, Sullivan SM, Neyrolles O, Wain JR, Patel J, Pool KL, Young DB, Wilkinson RJ (2005) Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis., Infect Immun 73 (10) pp. 6831-6837
The 19-kDa lipoprotein of Mycobacterium tuberculosis is an important target of the innate immune response. To investigate the immune biology of this antigen in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19-kDa-lipoprotein gene (Delta19) and complemented this strain by reintroduction of the 19-kDa-lipoprotein gene on a multicopy vector to produce Delta19::pSMT181. The Delta19 strain multiplied less well than Delta19::pSMT181 in human monocyte-derived macrophages (MDM) (P = 0.039). Surface expression of major histocompatibility complex class II molecules was reduced in phagocytes infected with M. tuberculosis; this effect was not seen in cells infected with Delta19. Delta19 induced lower interleukin 1beta (IL-1beta) secretion from monocytes and MDM. Overexpression of the 19-kDa protein increased IL-1beta, IL-12p40, and tumor necrosis factor alpha secretion irrespective of phagocyte maturity. These data support reports that the 19-kDa lipoprotein has pleiotropic effects on the interaction of M. tuberculosis with phagocytes. However, this analysis indicates that in the context of the whole bacillus, the 19-kDa lipoprotein is only one of a number of molecules that mediate the innate response to M. tuberculosis.
Mokhtar H, Biffar L, Somavarapu S, Frossard J, Mcgowan S, Pedrera M, Strong R, Edwards J, Garcia-Durán M, Rodriguez M, Stewart G, Steinbach F, Graham S (2017) Evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate T cell antigens, Veterinary Microbiology 209 pp. 66-74 Elsevier
PRRS control is hampered by the inadequacies of existing vaccines to combat the extreme diversity of circulating viruses. Since immune clearance of PRRSV infection may not be dependent on the development of neutralising antibodies and the identification of broadly- neutralising antibody epitopes have proven elusive we hypothesised that conserved T cell antigens represent potential candidates for development of a novel PRRS vaccine. Previously we had identified the M and NSP5 proteins as well-conserved targets of polyfunctional CD8 and CD4 T cells. To assess their vaccine potential, peptides representing M and NSP5 were encapsulated in hydrophobically-modified chitosan particles adjuvanted by incorporation of a synthetic multi-TLR2/TLR7 agonist and coated with a model B cell PRRSV antigen. For comparison, empty particles and adjuvanted particles encapsulating inactivated PRRSV-1
Butler Rachel, Krishnan N, Garcia-Jimenez W, Francis R, Martyn A, Mendum T, Felemban Shaza, Locker Nicolas, Salguero Bodes J, Robertson B, Stewart Graham (2017) Susceptibility of M. tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNF±, Virulence 8 (8) pp. 1820-1832 Taylor & Francis
An important feature of Mycobacterium tuberculosis pathogenesis is the ability to control
cell death in infected host cells, including inhibition of apoptosis and stimulation of necrosis.
Recently an alternative form of programmed cell death, necroptosis, has been described
where necrotic cell death is induced by apoptotic stimuli under conditions where apoptotic
execution is inhibited. We show for the first time that M. tuberculosis and TNF± synergise
to induce necroptosis in murine fibroblasts via RIPK1-dependent mechanisms and
characterized by phosphorylation of Ser345 of the MLKL necroptosis death effector.
However, in murine macrophages M. tuberculosis and TNF± induce non-necroptotic cell
death that is RIPK1-dependent but independent of MLKL phosphorylation. Instead, M.
tuberculosis-infected macrophages undergo RIPK3-dependent cell death which occurs both in the presence and absence of TNF± and involves the production of mitochondrial ROS.
Immunocytochemical staining for MLKL phosphorylation further demonstrated the
occurrence of necroptosis in vivo in murine M. tuberculosis granulomas. Phosphorylated-
MLKL immunoreactivity was observed associated with the cytoplasm and nucleus of
fusiform cells in M. tuberculosis lesions but not in proximal macrophages. Thus whereas
pMLKL-driven necroptosis does not appear to be a feature of M. tuberculosis-infected
macrophage cell death, it may contribute to TNF±-induced cytotoxicity of the lung stroma
and therefore contribute to necrotic cavitation and bacterial dissemination.
Donoghue H, Taylor G, Stewart G, Lee O, Wu H, Besra G, Minnikin D (2017) Positive Diagnosis of Ancient Leprosy and
Tuberculosis Using Ancient DNA and
Lipid Biomarkers,
Diversity 9 (4) 46 MDPI
Diagnosis of leprosy and tuberculosis in archaeological material is most informative when based upon entire genomes. Ancient DNA (aDNA) is often degraded but amplification of specific fragments also provides reliable diagnoses. Cell wall lipid biomarkers can distinguish ancient leprosy from tuberculosis and DNA extraction residues can be utilized. The diagnostic power of combined aDNA and lipid biomarkers is illustrated by key cases of ancient leprosy and/or tuberculosis. Human tuberculosis was demonstrated in a woman and child from Atlit-Yam (~9 ka) in the Eastern Mediterranean and in the 600 BCE Egyptian ?Granville? mummy. Both aDNA and lipids confirmed Pleistocene tuberculosis in a ~17 ka bison from Natural Trap Cave, Wyoming. Leprosy is exemplified by cases from Winchester (10th?12th centuries CE) and Great Chesterford (5th?6th centuries CE). A mixed infection from Kiskundorozsma, Hungary (7th century CE) allowed lipid biomarkers to assess the relative load of leprosy and tuberculosis. Essential protocols for aDNA amplification and analysis of mycolic, mycolipenic, mycocerosic acid, and phthiocerol lipid biomarkers are summarized. Diagnoses of ancient mycobacterial disease can be extended beyond the reach of whole genomics by combinations of aDNA amplification and lipid biomarkers, with sole use of the latter having the potential to recognize even older cases.
Inskip S, Taylor G, Anderson S, Stewart Graham (2017) Leprosy in pre-Norman Suffolk, UK: biomolecular and geochemical analysis of the woman from Hoxne, Journal of Medical Microbiology 66 pp. 1640-1649 Microbiology Society
Purpose.

A woman?s skull, exhibiting features of lepromatous leprosy (LL), was recovered from a garden in Hoxne, Suffolk. The absence of post crania and lack of formal excavation meant that diagnosis and dating was uncertain. The aim of this research was to confirm the diagnosis using biomolecular means and second, to place it in context with other British leprosy cases using SNP genotyping and radiocarbon dating.

Methodology.

Bone from the skull was analysed by ancient DNA (aDNA) methods and subjected to radiocarbon dating. As a result, stable carbon and nitrogen isotope values were produced, both useful for assessing aspects of the woman?s diet.

Results/Key findings.

aDNA confirmed the presence of mycobacterium leprae and genotyping demonstrated an ancestral variant of subtype 3I, the same lineage recently identified in living squirrels in the south of England. Radiocarbon dating revealed the woman lived approximately between 885?1015 AD, providing evidence for endurance of this subtype in East Anglia, having been previously identified as early as the fifth?sixth century (Great Chesterford) and as late as the thirteenth century (Ipswich).

Conclusions.

The confirmation of a new pre-Norman leprosy case in East Anglia is of interest as this is where a high proportion of cases are located. Possible factors for this may include preservation and excavation biases, population density, but also connection and trade, possibly of fur, with the continent. Future research on other British LL cases should focus on exploring these aspects to advance understanding of the disease?s history, here and on the continent.

Schuenemann Verena J, Avanzi Charlotte, Krause-Kyora Ben, Seitz Alexander, Herbig Alexander, Inskip Sarah, Bonazzi Marion, Reiter Ella, Urban Christian, Pedersen Dorthe Dangvard, Taylor G Michael, Singh Pushpendra, Stewart Graham, Veleminsky Petr, Likovsky Jakub, Marcsik Antonia, Molnar Erika, Palfi Gyorgy, Mariotti Valentina, Riga Alessandro, Belcastro M Giovanna, Boldsen Jesper L, Nebel Almut, Mays Simon, Donoghue Helen D, Zakrzewski Sonia, Benjak Andrej, Nieselt Kay, Cole Stewart T, Krause Johannes (2018) Ancient Mycobacterium leprae genomes reveal a high diversity of Mycobacterium leprae in medieval Europe, PLOS Pathogens 14 (5) e1006997 Public Library of Science
Studying ancient DNA allows us to retrace the evolutionary history of human pathogens,
such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the
oldest recorded and most stigmatizing diseases in human history. The disease was prevalent
in Europe until the 16th century and is still endemic in many countries with over 200,000
new cases reported annually. Previous worldwide studies on modern and European medieval
M. leprae genomes revealed that they cluster into several distinct branches of which two
were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval
M. leprae genomes including the so far oldest M. leprae genome from one of the earliest
known cases of leprosy in the United KingdomÐa skeleton from the Great Chesterford cemetery
with a calibrated age of 415±545 C.E. This dataset provides a genetic time transect of
M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four
distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether
these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at
various time points than previously assumed. The resulting more complex picture of the
past phylogeography of leprosy in Europe impacts current phylogeographical models of M.
leprae dissemination. It suggests alternative models for the past spread of leprosy such as a
wide spread prevalence of strains from different branches in Eurasia already in Antiquity or
maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying
ancient M. leprae strains improves understanding the history of leprosy worldwide.
Bucca Giselda, Pothi Radhika, Hesketh Andrew, Moller-Levet Carla, Hodgson David A, Laing Emma, Stewart Graham, Smith Colin (2018) Translational control plays an important role in the adaptive heat-shock response of Streptomyces coelicolor., Nucleic Acids Research 46 (11) pp. 5692-5703 Oxford University Press
Stress-induced adaptations requiremultiple levels of
regulation in all organisms to repair cellular damage.
In the present study we evaluated the genome-wide
transcriptional and translational changes following
heat stress exposure in the soil-dwelling model actinomycete
bacterium, Streptomyces coelicolor. The
combined analysis revealed an unprecedented level
of translational control of gene expression, deduced
through polysome profiling, in addition to transcriptional
changes. Our data show little correlation between
the transcriptome and ?translatome?; while an
obvious downward trend in genome wide transcription
was observed, polysome associated transcripts
following heat-shock showed an opposite upward
trend. A handful of key protein players, including
the major molecular chaperones and proteases were
highly induced at both the transcriptional and translational
level following heat-shock, a phenomenon
known as ?potentiation?. Many other transcripts encoding
cold-shock proteins, ABC-transporter systems,
multiple transcription factors weremore highly
polysome-associated following heat stress; interestingly,
these protein families were not induced at the
transcriptional level and therefore were not previously
identified as part of the stress response. Thus,
stress coping mechanisms at the level of gene expression
in this bacterium go well beyond the induction
of a relatively small number of molecular chaperones
and proteases in order to ensure cellular survival
at non-physiological temperatures.
Mendum Thomas, Taylor G Michael, Donoghue Helen D, Wu Huihai, Szalontai Csaba, Marcsik Antónia, Molnár Erika, Pálfi György, Stewart Graham (2018) The Genome Sequence of a SNP Type 3K strain of Mycobacterium leprae isolated from a 7th Century Hungarian case of Lepromatous Leprosy., International Journal of Osteoarchaeology 28 (4) pp. 439-447 Wiley
We report on a Mycobacterium leprae genome isolated from the remains of an individual with lepromatous leprosy that were excavated from a 7th century Hungarian cemetery. We determined that the genome was from a SNP type 3K0 M. leprae strain, a lineage that diverged early from other M. leprae lineages. This is one of the earliest 3K0 M. leprae genomes to be sequenced to date. A number of novel SNPs as well as SNPs characteristic of the 3K0 lineage were confirmed by conventional PCR and Sanger sequencing. Recovery of accompanying human DNA from the burial was poor, particularly when compared to that of the pathogen. Modern 3K0 M. leprae strains have only been isolated from East Asia and the Pacific and so these findings require new scenarios to describe the origins and routes of dissemination of leprosy during antiquity that have resulted in the modern phylogeographical distribution of M. leprae.