Genomic mapping of cAMP receptor protein (CRP Mt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor.

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRP(Mt)) at endogenous expression levels using a specific α-CRP(Mt) antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRP(Mt) binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRP(Mt)-binding site represented only a minor portion of this transcriptional reprogramming with ∼ 19% of those representing transcriptional regulators potentially controlled by CRP(Mt). The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRP(Mt) can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRP(Mt)-binding sites.

Effect of isoniazid on antigen-specific interferon-γ secretion in latent tuberculosis.

Treatment of persons with latent tuberculosis (TB) infection at greatest risk of reactivation is an important component of TB control and elimination strategies. Biomarkers evaluating the effectiveness of treatment of latent TB infection have not yet been identified. This information would enhance control efforts and assist the evaluation of new treatment regimes. We designed a two-group, two-arm, randomised clinical study of tuberculin skin test-positive participants: 26 with documented contact with TB patients and 34 with non-documented contact. Participants in each group were randomly assigned to the immediate- or deferred-isoniazid treatment arms. Assays of in vitro interferon (IFN)-γ secretion in response to recombinant Rv1737 and overlapping synthetic peptide pools from various groups of immunodominant proteins were performed. During isoniazid therapy, a significant increase from baseline in the proportion of IFN-γ responders to the 10-kDa culture filtrate protein, Rv2031, Rv0849, Rv1986, Rv2659c, Rv2693c and the recombinant Rv1737 protein was observed (p⩽0.05). The peptide pool of Rv0849 and Rv1737 recombinant proteins induced the highest percentage of IFN-γ responders after isoniazid therapy. The in vitro IFN-γ responses to these proteins might represent useful markers to evaluate changes associated with treatment of latent TB infection.

Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment.

Calcidiol, the major circulating metabolite of vitamin D, supports induction of pleiotropic antimicrobial responses in vitro. Vitamin D supplementation elevates circulating calcidiol concentrations, and thus has a potential role in the prevention and treatment of infection. The immunomodulatory effects of administering vitamin D to humans with an infectious disease have not previously been reported. To characterize these effects, we conducted a detailed longitudinal study of circulating and antigen-stimulated immune responses in ninety-five patients receiving antimicrobial therapy for pulmonary tuberculosis who were randomized to receive adjunctive high-dose vitamin D or placebo in a clinical trial, and who fulfilled criteria for per-protocol analysis. Vitamin D supplementation accelerated sputum smear conversion and enhanced treatment-induced resolution of lymphopaenia, monocytosis, hypercytokinaemia, and hyperchemokinaemia. Administration of vitamin D also suppressed antigen-stimulated proinflammatory cytokine responses, but attenuated the suppressive effect of antimicrobial therapy on antigen-stimulated secretion of IL-4, CC chemokine ligand 5, and IFN-α. We demonstrate a previously unappreciated role for vitamin D supplementation in accelerating resolution of inflammatory responses during tuberculosis treatment. Our findings suggest a potential role for adjunctive vitamin D supplementation in the treatment of pulmonary infections to accelerate resolution of inflammatory responses associated with increased risk of mortality.

Induced ectopic expression of HigB toxin in Mycobacterium tuberculosis results in growth inhibition, reduced abundance of a subset of mRNAs and cleavage of tmRNA.

In Mycobacterium tuberculosis, the genes Rv1954A-Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin-antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of the higBA locus, effects of toxin expression on mycobacterial growth and transcript levels were assessed in M. tuberculosis H37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death in M. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA in M. tuberculosis, Escherichia coli and Mycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB in M. tuberculosis.

Blockade of IL-10 signaling during bacillus Calmette-Guérin vaccination enhances and sustains Th1, Th17, and innate lymphoid IFN-γ and IL-17 responses and increases protection to Mycobacterium tuberculosis infection.

Vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) remains the only prophylactic vaccine against tuberculosis, caused by Mycobacterium tuberculosis, but gives variable protection against pulmonary disease. The generation of host Th1 responses following BCG vaccination is accepted as the major mechanism of protection against M. tuberculosis infection. Early production of IL-17 in the lungs following M. tuberculosis challenge of mice previously vaccinated with M. tuberculosis peptides in adjuvant has been shown to be required for efficient Th1 cell recruitment. IL-10 regulates various processes involved in generation of Th1 and Th17 responses. Previous studies have shown IL-10 as a negative regulator of the immune response to primary M. tuberculosis infection, with Il10(-/-) mice having reduced lung bacterial loads. In this study we show that inhibition of IL-10 signaling during BCG vaccination enhances host-generated Ag-specific IFN-γ and IL-17A responses, and that this regimen gives significantly greater protection against aerogenic M. tuberculosis challenge in both susceptible and relatively resistant strains of mice. In M. tuberculosis-susceptible CBA/J mice, Ab blockade of IL-10R specifically during BCG vaccination resulted in additional protection against M. tuberculosis challenge of >1-log(10) compared with equivalent isotype-treated controls. The protection observed following BCG vaccination concurrent with anti-IL-10R mAb treatment was sustained through chronic M. tuberculosis infection and correlated with enhanced lung Th1 and Th17 responses and increased IFN-γ and IL-17A production by γδ T cells and an innate-like Thy1.2(+)CD3(-) lymphoid population. We show that IL-10 inhibits optimal BCG-elicited protection, therefore suggesting that antagonists of IL-10 may be of great benefit as adjuvants in preventive vaccination against tuberculosis.

Bioinformatic and empirical analysis of novel hypoxia-inducible targets of the human antituberculosis T cell response.

We analyzed whole genome-based transcriptional profiles of Mycobacterium tuberculosis subjected to prolonged hypoxia to guide the discovery of novel potential Ags, by a combined bioinformatic and empirical approach. We analyzed the fold induction of the 100 most highly induced genes at 7 d of hypoxia, as well as transcript abundance, peptide-binding prediction (ProPred) adjusted for population-specific MHC class II allele frequency, and by literature search. Twenty-six candidate genes were selected by this bioinformatic approach and evaluated empirically using IFN-γ and IL-2 ELISPOT using immunodominant Ags (Acr-1, CFP-10, ESAT-6) as references. Twenty-three of twenty-six proteins induced an IFN-γ response in PBMCs of persons with active or latent tuberculosis. Five novel immunodominant proteins-Rv1957, Rv1954c, Rv1955, Rv2022c, and Rv1471-were identified that induced responses similar to CFP-10 and ESAT-6 in both magnitude and frequency. IL-2 responses were of lower magnitude than were those of IFN-γ. Only moderate evidence of infection stage-specific recognition of Ags was observed. Reconciliation of bioinformatic and empirical hierarchies of immunodominance revealed that Ags could be predicted, providing transcriptomic data were combined with peptide-binding prediction adjusted by population-specific MHC class II allele frequency.

Differential virulence and disease progression following Mycobacterium tuberculosis complex infection of the common marmoset (Callithrix jacchus).

Existing small-animal models of tuberculosis (TB) rarely develop cavitary disease, limiting their value for assessing the biology and dynamics of this highly important feature of human disease. To develop a smaller primate model with pathology similar to that seen in humans, we experimentally infected the common marmoset (Callithrix jacchus) with diverse strains of Mycobacterium tuberculosis of various pathogenic potentials. These included recent isolates of the modern Beijing lineage, the Euro-American X lineage, and M. africanum. All three strains produced fulminant disease in this animal with a spectrum of progression rates and clinical sequelae that could be monitored in real time using 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). Lesion pathology at sacrifice revealed the entire spectrum of lesions observed in human TB patients. The three strains produced different rates of progression to disease, various extents of extrapulmonary dissemination, and various degrees of cavitation. The majority of live births in this species are twins, and comparison of results from siblings with different infecting strains allowed us to establish that the infection was highly reproducible and that the differential virulence of strains was not simply host variation. Quantitative assessment of disease burden by FDG-PET/CT provided an accurate reflection of the pathology findings at necropsy. These results suggest that the marmoset offers an attractive small-animal model of human disease that recapitulates both the complex pathology and spectrum of disease observed in humans infected with various M. tuberculosis strain clades.

Mycobacterial lineages causing pulmonary and extrapulmonary tuberculosis, Ethiopia.

Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

A restatement of the natural science evidence base relevant to the control of bovine tuberculosis in Great Britain.

Bovine tuberculosis (bTB) is a very important disease of cattle in Great Britain, where it has been increasing in incidence and geographical distribution. In addition to cattle, it infects other species of domestic and wild animals, in particular the European badger (Meles meles). Policy to control bTB is vigorously debated and contentious because of its implications for the livestock industry and because some policy options involve culling badgers, the most important wildlife reservoir. This paper describes a project to provide a succinct summary of the natural science evidence base relevant to the control of bTB, couched in terms that are as policy-neutral as possible. Each evidence statement is placed into one of four categories describing the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Sequence-based analysis uncovers an abundance of non-coding RNA in the total transcriptome of Mycobacterium tuberculosis.

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5' and 3' untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.

OX40 ligand fusion protein delivered simultaneously with the BCG vaccine provides superior protection against murine Mycobacterium tuberculosis infection.

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.

Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

Confronting the scientific obstacles to global control of tuberculosis.

Tuberculosis (TB) is a major threat to global health, recently exacerbated by the emergence of highly drug-resistant forms of the disease-causing pathogen and synergy with HIV/AIDS. In 2006, the Stop TB Partnership published "The global plan to stop TB: 2006--2015," which set out a vision of halving the prevalence of and mortality caused by the disease by 2015, followed by eliminating the disease as a public health problem by 2050. This vision depends on the development of improved diagnostics, simpler treatment, and more effective vaccination. Recently, active translational research pipelines directed toward each of these goals have been established, but improved understanding of the fundamental biology of this complex disease will prove to be the key to radical advances in TB control.

Transcriptomic Characterization of Tuberculous Sputum Reveals a Host Warburg Effect and Microbial Cholesterol Catabolism.

The crucial transmission phase of tuberculosis (TB) relies on infectious sputum and yet cannot easily be modeled. We applied one-step RNA sequencing (RNA-Seq) to sputum from infectious TB patients to investigate the host and microbial environments underlying transmission of Mycobacterium tuberculosis. In such TB sputa, compared to non-TB controls, transcriptional upregulation of inflammatory responses, including an interferon-driven proinflammatory response and a metabolic shift toward glycolysis, was observed in the host. Among all bacterial sequences in the sputum, approximately 1.5% originated from M. tuberculosis, and its transcript abundance was lower in HIV-1-coinfected patients. Commensal bacterial abundance was reduced in the presence of M. tuberculosis infection. Direct alignment to the genomes of the predominant microbiota species also reveals differential adaptation, whereby firmicutes (e.g., streptococci) displayed a nonreplicating phenotype with reduced transcription of ribosomal proteins and reduced activities of ATP synthases, while Neisseria and Prevotella spp. were less affected. The transcriptome of sputum M. tuberculosis more closely resembled aerobic replication and shared similarity in carbon metabolism to in vitro and in vivo models with significant upregulation of genes associated with cholesterol metabolism and downstream propionate detoxification pathways. In addition, and counter to previous reports on intracellular M. tuberculosis infection in vitro, M. tuberculosis in sputum was zinc, but not iron, deprived, and the phoP loci were also significantly downregulated, suggesting that the pathogen is likely extracellular in location. IMPORTANCE Although a few studies have described the microbiome composition of TB sputa based on 16S ribosomal DNA, these studies did not compare to non-TB samples and the nature of the method does not allow any functional inference. This is the first study to apply such technology using clinical specimens and obtained functional transcriptional data on all three aspects simultaneously. We anticipate that an improved understanding on the biological interactions in the respiratory tract may also allow novel interventions, such as those involving microbiome manipulation or inhibitor targeting disease-specific metabolic pathways.

Identification of small RNAs in Mycobacterium tuberculosis.

In spite of being one of our most prominent bacterial pathogens, the presence of small regulatory RNAs (sRNAs) has not previously been investigated in Mycobacterium tuberculosis. Post-transcriptional regulation of gene expression by sRNA molecules has been demonstrated in a wide range of pathogenic bacteria and has been shown to play a significant role in the control of virulence. By screening cDNA libraries prepared from low-molecular weight RNA from M. tuberculosis we have identified nine putative sRNA molecules, including cis-encoded antisense transcripts from within open reading frames and trans-encoded transcripts from intergenic regions. sRNAs displayed differential expression between exponential and stationary phase, and during a variety of stress conditions. Two of the cis-encoded sRNAs were associated with genes encoding enzymes involved in lipid metabolism, desA1 and pks12. These sRNAs showed complementarity to multiple M. tuberculosis genes, suggesting the potential to act as both cis-encoded and trans-encoded sRNAs. Overexpression of selected trans-encoded sRNAs had profound impact on growth of M. tuberculosis and M. smegmatis. This is the first experimental evidence of sRNAs in M. tuberculosis and it will be important to consider the potential influence of sRNA regulation when studying the transcriptome and the proteome of M. tuberculosis during infection.

Genetic determination of the effect of post-translational modification on the innate immune response to the 19 kDa lipoprotein of Mycobacterium tuberculosis.

BACKGROUND: The 19 kDa lipoprotein of Mycobacterium tuberculosis (MTB) is an important target of the innate immune response. To investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant M. tuberculosis H37Rv that lacked the 19 kDa gene (Delta19) and complemented this strain by reintroduction of the 19 kDa gene into the chromosome as a single copy to produce Delta19::19. We also reintroduced the 19 kDa gene in two modified forms that lacked motifs for acylation (Delta19::19NA) and O-glycosylation (Delta19::19NOG). RESULTS: Both acylation and O-glycosylation were necessary for the protein to remain within the cell. IL-1 Beta secretion from human monocytes was significantly reduced by deletion of the 19 kDa gene (p < 0.02). Complementation by the wild type, but not the mutagenised gene reversed this phenotype. The effect of deletion and complementation on IL-12p40 and TNF secretion was less marked with no statistically significant differences between strains. Although deletion of the 19 kDa reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not achieve statistical significance. CONCLUSION: These results confirm in the context of the whole bacillus an important role for post-translational modification of the 19 kDa on both the cellular location and immune response to this protein.

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

Heat-shock proteins and the host-pathogen interaction during bacterial infection.

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.

Unusual features of the cell cycle in mycobacteria: polar-restricted growth and the snapping-model of cell division.

Cell division patterns in mycobacteria have been examined in order to further our understanding of how these important organisms grow in the apparent absence of key systems required for the growth of rod-shaped bacteria. Analysis of the distribution of cell lengths in the population during different phases of growth showed that the modal cell length decreases during later phases of growth, declining from 3.5 to 2.5 microm for Mycobacterium bovis BCG cells sampled in log phase and stationary phase, respectively. The population also became more homogeneous, as indicated by the proportion of cells in the most common class increasing from 15% to 28%. Similar patterns were observed for Mycobacterium smegmatis and Mycobacterium tuberculosis. Consistent with other actinomycetes, and in contrast to most rod-shaped bacteria, the deposition of newly synthesised peptidoglycan in mycobacteria is restricted to the poles of the cell, as evidenced by staining with fluorescently labelled vancomycin. A "V-form" of bacteria was observed in cultures at all stages of growth, but the proportion decreased in older cultures. The V-shape appears to be a result of the uneven splitting of the exterior cell envelope at the new septum; this exposes the new peptidoglycan which is illustrated by spots of fluorescent vancomycin staining associated with the exterior side of the "V", and supports the 'snapping division model'. The restriction of growth to the poles of the cell differs from the pattern observed in other rod-shaped bacteria, in which the cell poles are inert and lateral growth occurs by deposition of peptidoglycan along the body of the cylinder. The mechanisms that maintain the shape of mycobacteria and that identify the mid-point for cell division remain to be determined.