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.
KA Wilkinson, SM Newton, GR Stewart, AR Martineau, J Patel, SM Sullivan, J-L Herrmann, O Neyrolles, DB Young, RJ Wilkinson (2009)Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis, In: BMC MICROBIOLOGY9ARTN 9pp. ?-?
BIOMED CENTRAL LTD
GR Stewart, KA Wilkinson, SA Newton, SA Sullivan, O Neyrolles, JR Wain, J Patel, KL Pool, DB Young, RJ Wilkinson (2005)Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis, In: INFECTION AND IMMUNITY73(10)pp. 6831-6837
AMER SOC MICROBIOLOGY
DB Young, GR Stewart (2002)Tuberculosis vaccines, In: BRITISH MEDICAL BULLETIN62pp. 73-86
OXFORD UNIV PRESS
P Brodin, Y Poquet, F Levillain, I Peguillet, G Larrouy-Maumus, M Gilleron, F Ewann, T Christophe, D Fenistein, J Jang, M-S Jang, S-J Park, J Rauzier, J-P Carralot, R Shrimpton, A Genovesio, JA Gonzalo-Asensio, G Puzo, C Martin, R Brosch, GR Stewart, B Gicquel, O Neyrolles (2010)High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling, In: PLOS PATHOGENS6(9)ARTN epp. ?-?
PUBLIC LIBRARY SCIENCE
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.
G. Michael Taylor, Eileen M. Murphy, Thomas Mendum, Alistair W.G Pike, Bethan Linscott, Huihai Wu, Justin O'Grady, Hollian Richardson, Edmond O'Donovan, Carmelita Troy, Graham Stewart (2018)Leprosy at the edge of Europe—Biomolecular, isotopic and osteoarchaeological findings from medieval Ireland, In: PLOS One
Public Library of Science
Relatively little is known of leprosy in Medieval Ireland; as an island located at the far west of Europe it has the potential to provide interesting insights in relation to the historical epidemiology of the disease. To this end the study focuses on five cases of probable leprosy identified in human skeletal remains excavated from inhumation burials. Three of the individuals derived from the cemetery of St Michael Le Pole, Golden Lane, Dublin, while single examples were also identified from Ardreigh, Co. Kildare, and St Patrick’s Church, Armoy, Co. Antrim. The individuals were radiocarbon dated and examined biomolecularly for evidence of either of the causative pathogens, M. leprae or M. lepromatosis. Oxygen and strontium isotopes were measured in tooth enamel and rib samples to determine where the individuals had spent their formative years and to ascertain if they had undertaken any recent migrations. We detected M. leprae DNA in the three Golden Lane cases but not in the probable cases from either Ardreigh Co. Kildare or Armoy, Co. Antrim. M. lepromatosis was not detected in any of the burals. DNA preservation was sufficiently robust to allow genotyping of M. leprae strains in two of the Golden Lane burials, SkCXCV (12-13th century) and SkCCXXX (11-13th century). These strains were found to belong on different lineages of the M. leprae phylogenetic tree, namely branches 3 and 2 respectively. Whole genome sequencing was also attempted on these two isolates with a view to gaining further information but poor genome coverage precluded phylogenetic analysis. Data from the biomolecular study was combined with osteological, isotopic and radiocarbon dating to provide a comprehensive and multidisciplinary study of the Irish cases. Strontium and oxygen isotopic analysis indicate that two of the individuals from Golden Lane (SkCXLVIII (10-11th century) and SkCXCV) were of Scandinavian origin, while SkCCXXX may have spent his childhood in the north of Ireland or central Britain. We propose that the Vikings were responsible for introducing leprosy to Ireland. This work adds to our knowledge of the likely origins of leprosy in Medieval Ireland and will hopefully stimulate further research into the history and spread of this ancient disease across the world.
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.
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.
H Mokhtar, L Biffar, S Somavarapu, J-P Frossard, Sarah Mcgowan, M Pedrera, R Strong, JC Edwards, M Garcia-Durán, MJ Rodriguez, Graham Stewart, Falko Steinbach, Simon Graham (2017)Evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate T cell antigens, In: Veterinary Microbiology209pp. 66-74
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
Mycobacterium tuberculosis infects a third of the world's population. Primary tuberculosis involving active fast bacterial replication is often followed by asymptomatic latent tuberculosis, which is characterised by slow or non-replicating bacteria. Reactivation of the latent infection involving a switch back to active bacterial replication can lead to post-primary transmissible tuberculosis. Mycobacterial mechanisms involved in slow growth or switching growth rate provide rational targets for the development of new drugs against persistent mycobacterial infection. Using chemostat culture to control growth rate, we screened a transposon mutant library by Transposon site hybridization (TraSH) selection to define the genetic requirements for slow and fast growth of Mycobacterium bovis (BCG) and for the requirements of switching growth rate. We identified 84 genes that are exclusively required for slow growth (69 hours doubling time) and 256 genes required for switching from slow to fast growth. To validate these findings we performed experiments using individual M. tuberculosis and M. bovis BCG knock out mutants. We have demonstrated that growth rate control is a carefully orchestrated process which requires a distinct set of genes encoding several virulence determinants, gene regulators, and metabolic enzymes. The mce1 locus appears to be a component of the switch to slow growth rate, which is consistent with the proposed role in virulence of M. tuberculosis. These results suggest novel perspectives for unravelling the mechanisms involved in the switch between acute and persistent TB infections and provide a means to study aspects of this important phenomenon in vitro.
GR Stewart, VA Snewin, G Walzl, T Hussell, P Tormay, P O'Gaora, M Goyal, J Betts, IN Brown, DB Young (2001)Overexpression of heat-shock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection., In: Nat Med7(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.
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.
Verena J Schuenemann, Charlotte Avanzi, Ben Krause-Kyora, Alexander Seitz, Alexander Herbig, Sarah Inskip, Marion Bonazzi, Ella Reiter, Christian Urban, Dorthe Dangvard Pedersen, G Michael Taylor, Pushpendra Singh, Graham Stewart, Petr Veleminsky, Jakub Likovsky, Antonia Marcsik, Erika Molnar, Gyorgy Palfi, Valentina Mariotti, Alessandro Riga, M Giovanna Belcastro, Jesper L Boldsen, Almut Nebel, Simon Mays, Helen D Donoghue, Sonia Zakrzewski, Andrej Benjak, Kay Nieselt, Stewart T Cole, Johannes Krause (2018)Ancient Mycobacterium leprae genomes reveal a high diversity of Mycobacterium leprae in medieval Europe, In: PLOS Pathogens14(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.
DJV Beste, T Hooper, G Stewart, B Bonde, C Avignone-Rossa, M Bushell, P Wheeler, S Klamt, AM Kierzek, J McFadden (2007)GSMN-TB: a web-based genome scale network model of Mycobacterium tuberculosis metabolism, In: GENOME BIOLOGY8(5)ARTN rpp. ?-?
BIOMED CENTRAL LTD
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.
Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.
Rachel Butler, N Krishnan, W Garcia-Jimenez, R Francis, A Martyn, T Mendum, Shaza Felemban, Nicolas Locker, J Salguero Bodes, B Robertson, Graham Stewart (2017)Susceptibility of M. tuberculosis-infected host cells to phospho-MLKL driven necroptosis is dependent on cell type and presence of TNFα, In: Virulence8(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 TNFα 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 TNFα 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 TNFα 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 TNFα-induced cytotoxicity of the lung stroma and therefore contribute to necrotic cavitation and bacterial dissemination.
TA Mendum, VJ Schuenemann, S Roffey, GM Taylor, H Wu, P Singh, K Tucker, J Hinds, ST Cole, AM Kierzek, K Nieselt, J Krause, GR Stewart (2014)Mycobacterium leprae genomes from a British medieval leprosy hospital: towards understanding an ancient epidemic, In: BMC GENOMICS15ARTN 2pp. ?-?
BIOMED CENTRAL LTD
Mycobacterium bovis is the causative agent of bovine tuberculosis and the predominant cause of zoonotic tuberculosis in people. Bovine tuberculosis occurs in farmed cattle but also in a variety of wild animals, which form a reservoir of infection. Although direct transmission of tuberculosis occurs between mammals, the low frequency of contact between different host species and abundant shedding of bacilli by infected animals suggests an infectious route via environmental contamination. Other intracellular pathogens that transmit via the environment deploy strategies to survive or exploit predation by environmental amoebae. To explore if M. bovis has this capability, we investigated its interactions with the soil and dung-dwelling amoeba, Dictyostelium discoideum. We demonstrated that M. bovis evades phagocytosis and destruction by D. discoideum and actively transits through the amoeba using the ESX-1 Type VII Secretion System as part of a programme of mechanisms, many of which have been co-opted as virulence factors in the mammalian host. This capacity of M. bovis to utilise an environmental stage between mammalian hosts may enhance its transmissibility. In addition, our data provide molecular evidence to support an evolutionary role for amoebae as training grounds for the pathogenic M. tuberculosis complex.
DE Minnikin, GM Taylor, GR Stewart, TA Mendum, S Roffey, K Tucker, OY-C Lee, HHT Wu, GS Besra, ST Cole (2014)Defining the origins and spread of leprosy using molecular biomarkers, In: AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY153pp. 186-187 Tom A. Mendum, Aneesh Chandran, Kerstin Williams, H. Martin Vordermeier, Bernardo Villarreal-Ramos, H. Wu, Albel Singh, Alex A. Smith, Rachel E. Butler, Aravind Prasad, Neeraj Bharti, Ruma Banerjee, Sunitha M. Kasibhatla, Apoorva Bhatt, Graham R. Stewart, Johnjoe McFadden (2019)Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle, In: BMC Genomics20431pp. 1-13
BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG’s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host’s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria.
To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host.
These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host’s innate immune system to allow it to persist, a property that is important for its protective efficacy.
SM Newton, RJ Smith, KA Wilkinson, MP Nicol, NJ Garton, KJ Staples, GR Stewart, JR Wain, AR Martineau, S Fandrich, T Smallie, B Foxwell, A Al-Obaidi, J Shafi, K Rajakumar, B Kampmann, PW Andrew, L Ziegler-Heitbrock, MR Barer, RJ Wilkinson (2006)A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion, In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA103(42)pp. 15594-15598
NATL ACAD SCIENCES
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.
S Pitarque, JL Herrmann, JL Duteyrat, M Jackson, GR Stewart, F Lecointe, B Payre, O Schwartz, DB Young, G Marchal, PH Lagrange, G Puzo, B Gicquel, J Nigou, O Neyrolles (2005)Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity, In: BIOCHEMICAL JOURNAL392pp. 615-624
PORTLAND PRESS LTD
A Ciaramella, A Cavone, MB Santucci, SK Garg, N Sanarico, M Bocchino, D Galati, A Martino, G Auricchio, M D'Orazio, GR Stewart, O Neyrolles, DB Young, V Colizzi, M Fraziano (2004)Induction of apoptosis and release of interleukin-1 beta by cell wall-associated 19-kDa lipoprotein during the course of mycobacterial infection, In: JOURNAL OF INFECTIOUS DISEASES190(6)pp. 1167-1176 JE BRADLEY, L ELSON, TIM TREE, G STEWART, R GUDERIAN, M CALVOPINA, W PAREDES, E ARAUJO, TB NUTMAN (1995)RESISTANCE TO ONCHOCERCA-VOLVULUS - DIFFERENTIAL CELLULAR AND HUMORAL RESPONSES TO A RECOMBINANT ANTIGEN, OVMBP20/11, In: JOURNAL OF INFECTIOUS DISEASES172(3)pp. 831-837
UNIV CHICAGO PRESS
Huihai Wu, A von Kamp, Vytautas Leoncikas, W Mori, N Sahin, A Gevorgyan, C Linley, M Grabowski, AA Mannan, Nicholas Stoy, Graham Stewart, LT Ward, David Lewis, J Sroka, H Matsuno, S Klamt, HV Westerhoff, Johnjoe McFadden, NJ Plant, Andrzej Kierzek (2016)MUFINS: Multi-Formalism Interaction Network Simulator, In: NPJ Systems Biology and Applications216032
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.
Bovine tuberculosis is of huge animal health and economic concern in the UK. Current ‘test and slaughter’ control measures are not sufficient at controlling the disease, with evidence suggesting the introduction of an efficacious cattle vaccine being a viable alternative to reduce annual herd incidence. Approaches using the human tuberculosis vaccine, the live attenuated Mycobacterium bovis strain Bacillus Calmette–Guérin (BCG) Danish, as part of a prime/boost strategy have been shown to be effective at inducing immunity in cattle. One of the most effective vaccination strategies involves the development of a subunit vaccine for use alongside BCG. To this end, identification of protective antigens for use in a subunit vaccine is key. Here we describe a BCG transposon library mutant selection in cattle to identify the protective antigens of BCG. Prior to the cattle screen, the ‘selectability’ of the transposon library was validated using the mycobacterial heat-shock response and isoniazid resistance mechanisms. The preliminary screens identified essential genes in the respective pathways, such as hspR and katG, validating the use of the library to select for genes relevant to a selection pressure. Following validation, BCG vaccinated and immunologically naïve cattle were intranodally challenged with the transposon library and transposon mutants with comparatively better survival in BCG vaccinated animals were identified using TnSeq. The screen identified Mb0025, Mb0086, YrbE1B, PPE20 and Mb2235 as potential antigenic proteins of BCG. Mb0025 and Mb2235 were recombinantly expressed using an E. coli expression system. Mb0086, YrbE1B and PPE20 were purchased as peptides. The immunogenicity of the selected proteins was assessed in PBMCs harvested from immunologically naïve and BCG vaccinated cattle. IFN-γ, IP10, IL-β, IL-17a, IL-10 and IL-12 release from simulated PBMCs was quantified. IP10 production significantly increased upon stimulation with Mb0086, YrbE1B and PPE20 in vaccinated animals, with a trend of increased IL-17a and IL-10. Mb0086 and YrbE1B stimulated an increase in IL-1β production. Although protection has not been proven, this study has acted as a gate to select the best possible candidates for further experimentation in small animal studies. The transposon library screen also identified genes encoding enzymes involved in the synthesis of glycolipid antigens, such as ppm1 and ltp2, suggesting the inclusion of immunogenic glycolipids in a subunit vaccine along with protein antigens as an area for further study.
The human bacterial pathogen Neisseria meningitidis (Nm) is one of the world’s leading causative agents of systemic meningitis, which is characterised by inflammation of the meninges and more rarely, septicaemia. The high mortality rate and debilitating long term effects found in infants and young adults is due to the mounting of a large scale immune response by the host leading to severe inflammation and tissue death. The reasons as to how the meningococcus is able to breach innate immune barriers to cause this kind of infection, however, is still unclear. One important innate immune defence mechanism is the production of acute phase proteins such as C-Reactive Protein (CRP), which is known to opsonise pathogens such as N. meningitidis and enhance its uptake into human phagocytic cells. This investigation has demonstrated the enhanced uptake of CRP opsonised Nm into human macrophages and dendritic cells (DCs); two important innate immune cells which are some of the first to encounter the bacterium upon its dissemination through mucosal epithelium into the blood stream. Not only are these cells important phagocytes needed for the clearance of this pathogen, but dendritic cells in particular are the crucial link to the adaptive immune system, and play an important role in mounting effective, long term immunity to pathogens such as Nm. The enhanced uptake of CRP opsonised Nm was shown to occur by Fc gamma (γ) receptors I and II, which are expressed by both macrophages and dendritic cells. This was demonstrated by the blocking of Fcγ receptors with human IgG and specific antibodies to either receptor, which significantly reduced CRP enhanced uptake. The effect of CRP enhanced uptake of Nm into DC downstream immune responses was investigated to assess if CRP exacerbates or abrogates the hosts inflammatory response to Nm. Both unopsonised and CRP opsonised Nm were able to activate DCs and stimulate the up regulation of co-stimulatory and antigen presentation molecules, which is of significant importance in order to present processed Nm antigens to T cells and mount effective adaptive immune responses. Additionally, unopsonised Nm and CRP opsonised Nm were equally capable of up regulating the secretion of inflammatory cytokines from DCs. This suggests that CRP enhanced uptake of Nm by these phagocytic cells may be beneficial towards the host as despite the increased uptake of Nm by both cell types (potentially for phagocytic clearance), the pro-inflammatory cytokine response by DCs is not further exacerbated, thereby limiting inflammatory damage, which is known to the be the major contributor to the pathogenesis of meningococcal meningitis.
Human norovirus (HuNV) is a member of the calicivirus family and is a major cause of viral gastroenteritis worldwide. Due to the absence of a suitable cell culture system, HuNoV replication mechanisms are poorly understood, but two animal caliciviruses, Feline calicivirus (FCV) and Murine Norovirus (MNV) provide models to increase our understanding of norovirus biology. Unlike cellular mRNAs, the calicivirus RNA genome does not possess a 5' cap structure but instead has a 13–15 kDa viral protein, genome linked (VPg) directing translation, hijacking the host protein synthesis machinery. The viral life cycle requires separated events occurring at different times since viral transcripts are used as the template both for translation (mRNA) and replication (genomic RNA). Therefore mechanisms are required to control the viral RNA fate. In eukaryotes, during stress conditions, mRNAs can be stored in subcellular compartments such as stress granules to stall their translation or in processing bodies to be degraded. Recent evidence indicates that these compartments also play an important role during the viral life cycle. Therefore, using immunofluorescence microscopy we set out to investigate how FCV and MNV infection regulate the formation of G3BP1- and PABP-1-containing stress granules and DCP-1-containing processing bodies to address whether these cytoplasmic granules could play a role during the viral life cycle. We have now shown that FCV has the ability to prevent stress granules formation during infection and that this is important for replication in CRFK and FEA cells. Using FCV-free supernatant from infected CRFK cells and immunofluorescence microscopy, we have also shown that during infection, the formation of stress granules is induced in a paracrine manner in uninfected cells via a messenger molecule released from infected cells. We hypothesize that this could reflect a new antiviral role for stress granules. Furthermore, MNV and FCV infection also led to the disruption of processing-bodies assembly. Overall, this study revealed that caliciviruses modulate the RNA granules during infection and that this could be part of viral mechanism to counteract the antiviral response.
Streptomyces coelicolor and Mycobacterium bovis are high G+C gram-positive members of the phylum Actinobacteria. M. bovis is a member of the M. tuberculosis species complex and M. bovis, BCG is the attenuated vaccine strain used as a model organism as it can be manipulated in containment Category 2 laboratories. When cells are exposed to different environments/stresses they need to adapt their physiology and biochemistry for their effective survival. A key mechanism of adaptation is the ability to quickly alter the molecular composition of the cell through the regulation of gene expression both at transcriptional and translational level. While transcriptional regulation of gene expression has been studied extensively, little information regarding translational regulation in bacteria is available. To begin to study translational regulation in Streptomyces and Mycobacterium, a genome-wide approach was adapted to study the extent of translational regulation when cells are exposed to heat shock. As an initial attempt the protocol to isolate polysomes from both S. coelicolor and M. bovis (BCG) using classical sucrose density gradient under normal and heat shock conditions was optimized. Using DNA microarrays and RNA sequencing, the relative distribution of mRNA in the polysome fractions were analysed and compared it to the total transcriptome of the cell. Major heat shock responsive genes such as dnaK, grpE, groEL, groES, lon, hspR and dnaJ were significantly differentially expressed or were found to be translationally/transcriptionally potentiated (significantly differentially expressed genes in both transcription and translation) during heat shock in S. coelicolor, and in M. bovis (BCG) a smaller number of genesincluding SerX coding for tRNA biosynthesis and an ArsR repressor anti-toxin coding gene were shown to be translationally regulated. The study was further extended in S. coelicolor to investigate the use of a method based on the purification of affinity tagged ribosomes as a way to isolate actively translated mRNA. Selected ribosomal proteins were successfully tagged using strep-tag and the presence of tagged ribosomal proteins in cell lysates was checked using blotting techniques.
Tuberculosis (TB) is a major health problem worldwide resulting in 1.4 million deaths, caused by Mycobacterium tuberculosis. Despite all the efforts to target and eliminate this chronic disease, the control of tuberculosis has been severely thwarted by the emergence of multidrug and extensively resistant strains. The long treatment duration and its association with various side effects result in noncompliance of the patients. To improve treatment outcomes and reduce duration of therapy, host-directed TB therapies could provide a solution for the resolution of the disease. The development of host directed therapies will be expedited by further understanding of host-mycobacterium interaction and how the pathogen hijacks host cell processes to facilitate survival. Key to this process is the regulation of host gene expression. However, very little is known about translational control by bacterial pathogens, including Mycobacterium tuberculosis and how this contributes to pathogenesis. By using Mycobacterium bovis Bacillus Calmette-Guerin (BCG) as a surrogate of Mycobacterium.tuberculosis, we aimed to dissect how Mycobacterium bovis BCG alters translation in the infected macrophages, and how the regulation of eIF4E activity participates in this response to infection. Our results suggest that mycobacterial infection induces eIF4E phosphorylation in murine macrophages. Furthermore, the kinases ERK and MNK are responsible for eIF4E phosphorylation and their activation contributes to changes in the translational state of host mRNAs, as identified by polysome profiling. These changes alter the macrophage response to mycobacteria, affecting intracellular bacterial survival and macrophage viability. As it is believed that in up to 50 % of TB exposed individuals, the infection is cleared without the involvement of the adaptive immune system, indicating that the innate immune system may be able to control or clear the infection if activated appropriately. Further testing of the mechanisms used by macrophages to keep the infection under control has been done by measuring TNFα and IL-10 production, phagosomal acidity and cellular autophagy in the presence of ERK and MNK inhibitors. We found that the activation of ERK-MNK-eIF4E pathway regulates the cytokines production, but only ERK plays a regulatory role on macrophage phagosomal acidification as well as cell autophagy. Our finding suggests that Mycobacterium bovis BCG benefits from the activated ERK-MNK- eIF4E signalling to survive inside the cell. We conclude that regulating eIF4E phosphorylation is a key component of the hostpathogen interaction during mycobacterium infection and therefore, we suggest the possibility of using selective MNK and/or ERK inhibitors as host-directed immuno-therapeutics for tuberculosis.
Porcine reproductive and respiratory syndrome (PRRS) is one of the most important pig diseases worldwide. The causative PRRS virus (PRRSV) is rapidly evolving and there is an urgent need for the development of safer and more efficacious vaccines to improve PRRS control. Immunity to PRRSV is not well understood but there are data suggesting that virus-specific T cell IFN-γ responses play an important role. Therefore, this project focussed on characterising the T cell response PRRSV and applied this knowledge to develop a novel vaccine strategy. The first part of the project aimed to identify the antigenic targets of the T cell response to PRRSV by utilising a proteome-wide synthetic peptide library and a cohort of PRRSV immune animals. The T cell IFN-γ response was directed at a range of viral proteins but the M and NSP5 proteins stood out as major antigens. Further experiments confirmed M and NSP5 as well conserved targets of in many instances dominant T cell responses. Characterisation of the responding T cell populations showed NSP5-specific responders to be CD8 T cells with a predominant CD44highCD62LlowCD27lowCD25- phenotype. The majority of cells were polyfunctional as assessed by co-expression of TNF-α and mobilisation of the cytotoxic degranulation marker CD107a. Both CD8 and CD4 T cells responded to M with a comparable phenotype to that observed for NSP-specific T cells. In addition, conserved antigenic regions of each protein were identified and specificity shown to associate with major histocompatibility complex haplotype, rather than PRRSV strain. Finally, a vaccine study was conducted using M and NSP5 proteins as T cell antigens formulated as a particulate vaccine with a molecular adjuvant. Vaccination primed antigen-specific CD4 but not CD8 T cell responses and did not confer significant protection of animals from viraemia upon challenge infection. Analysis of the lungs during the resolution of infection showed high levels of virus and M/NSP5 specific CD8 T cell IFN-γ responses, suggesting that vaccine priming of a CD8 T cell response is required for protection from PRRSV infection. It is hoped that this work will inform future PRRSV vaccine design, as well as contributing to the wider field of T cell vaccinology.