Inhibition of Respiration by Nitric Oxide Induces a Mycobacterium tuberculosis Dormancy Program

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis

Comparative analysis of mycobacterium and related actinomycetes yields insight into the evolution of mycobacterium tuberculosis pathogenesis

<p>Abstract</p> <p>Background</p> <p>The sequence of the pathogen <it>Mycobacterium tuberculosis </it>(<it>Mtb</it>) strain <it>H37Rv </it>has been available for over a decade, but the biology of the pathogen remains poorly understood. Genome sequences from other <it>Mtb </it>strains and closely related bacteria present an opportunity to apply the power of comparative genomics to understand the evolution of <it>Mtb </it>pathogenesis. We conducted a comparative analysis using 31 genomes from the Tuberculosis Database (TBDB.org), including 8 strains of <it>Mtb </it>and <it>M. bovis</it>, 11 additional Mycobacteria, 4 Corynebacteria, 2 Streptomyces, <it>Rhodococcus jostii RHA1, Nocardia farcinia, Acidothermus cellulolyticus, Rhodobacter sphaeroides, Propionibacterium acnes</it>, and <it>Bifidobacterium longum</it>.</p> <p>Results</p> <p>Our results highlight the functional importance of lipid metabolism and its regulation, and reveal variation between the evolutionary profiles of genes implicated in saturated and unsaturated fatty acid metabolism. It also suggests that DNA repair and molybdopterin cofactors are important in pathogenic Mycobacteria. By analyzing sequence conservation and gene expression data, we identify nearly 400 conserved noncoding regions. These include 37 predicted promoter regulatory motifs, of which 14 correspond to previously validated motifs, as well as 50 potential noncoding RNAs, of which we experimentally confirm the expression of four.</p> <p>Conclusions</p> <p>Our analysis of protein evolution highlights gene families that are associated with the adaptation of environmental Mycobacteria to obligate pathogenesis. These families include fatty acid metabolism, DNA repair, and molybdopterin biosynthesis. Our analysis reinforces recent findings suggesting that small noncoding RNAs are more common in Mycobacteria than previously expected. Our data provide a foundation for understanding the genome and biology of <it>Mtb </it>in a comparative context, and are available online and through TBDB.org.</p

Sputum is a surrogate for bronchoalveolar lavage for monitoring Mycobacterium tuberculosis transcriptional profiles in TB patients

SummaryPathogen-targeted transcriptional profiling in human sputum may elucidate the physiologic state of Mycobacterium tuberculosis (M. tuberculosis) during infection and treatment. However, whether M. tuberculosis transcription in sputum recapitulates transcription in the lung is uncertain. We therefore compared M. tuberculosis transcription in human sputum and bronchoalveolar lavage (BAL) samples from 11 HIV-negative South African patients with pulmonary tuberculosis. We additionally compared these clinical samples with in vitro log phase aerobic growth and hypoxic non-replicating persistence (NRP-2). Of 2179 M. tuberculosis transcripts assayed in sputum and BAL via multiplex RT-PCR, 194 (8.9%) had a p-value <0.05, but none were significant after correction for multiple testing. Categorical enrichment analysis indicated that expression of the hypoxia-responsive DosR regulon was higher in BAL than in sputum. M. tuberculosis transcription in BAL and sputum was distinct from both aerobic growth and NRP-2, with a range of 396–1020 transcripts significantly differentially expressed after multiple testing correction. Collectively, our results indicate that M. tuberculosis transcription in sputum approximates M. tuberculosis transcription in the lung. Minor differences between M. tuberculosis transcription in BAL and sputum suggested lower oxygen concentrations or higher nitric oxide concentrations in BAL. M. tuberculosis-targeted transcriptional profiling of sputa may be a powerful tool for understanding M. tuberculosis pathogenesis and monitoring treatment responses in vivo

Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing

Increased DNA microarray hybridization specificity using sscDNA targets

Abstract Background The most widely used amplification method for microarray analysis of gene expression uses T7 RNA polymerase-driven in vitro transcription (IVT) to produce complementary RNA (cRNA) that can be hybridized to arrays. However, multiple rounds of amplification are required when assaying very small amounts of starting RNA. Moreover, certain cRNA-DNA mismatches are more stable than the analogous cDNA-DNA mismatches and this might increase non-specific hybridization. We sought to determine whether a recently developed linear isothermal amplification method (ribo-SPIA) that produces single stranded cDNA would offer advantages over traditional IVT-based methods for microarray-based analyses of transcript expression. Results A single round of ribo-SPIA amplification produced sufficient sscDNA for hybridizations when as little as 5 ng of starting total RNA was used. Comparisons of probe set signal intensities obtained from replicate amplifications showed consistently high correlations (r = 0.99). We compared gene expression in two different human RNA samples using ribo-SPIA. Compared with one round IVT, ribo-SPIA had a larger dynamic range and correlated better with quantitative PCR results even though we used 1000-fold less starting RNA. The improved dynamic range was associated with decreases in hybridization to mismatch control probes. Conclusion The use of amplified sscDNA may offer substantial advantages over IVT-based amplification methods, especially when very limited amounts of starting RNA are available. The use of sscDNA targets instead of cRNA targets appears to improve hybridization specificity.</p

New Genome-Wide Algorithm Identifies Novel In-Vivo Expressed Mycobacterium Tuberculosis Antigens Inducing Human T-Cell Responses with Classical and Unconventional Cytokine Profiles

16 páginas, 8 figuras. Disponoble información suplementaria en: http://www.nature.com/srepNew strategies are needed to develop better tools to control TB, including identification of novel antigens for vaccination. Such Mtb antigens must be expressed during Mtb infection in the major target organ, the lung, and must be capable of eliciting human immune responses. Using genome-wide transcriptomics of Mtb infected lungs we developed data sets and methods to identify IVE-TB (in-vivo expressed Mtb) antigens expressed in the lung. Quantitative expression analysis of 2,068 Mtb genes from the predicted first operons identified the most upregulated IVE-TB genes during in-vivo pulmonary infection. By further analysing high-level conservation among whole-genome sequenced Mtb-complex strains (n = 219) and algorithms predicting HLA-class-Ia and II presented epitopes, we selected the most promising IVE-TB candidate antigens. Several of these were recognized by T-cells from in-vitro Mtb-PPD and ESAT6/CFP10-positive donors by proliferation and multi-cytokine production. This was validated in an independent cohort of latently Mtb-infected individuals. Significant T-cell responses were observed in the absence of IFN-γ-production. Collectively, the results underscore the power of our novel antigen discovery approach in identifying Mtb antigens, including those that induce unconventional T-cell responses, which may provide important novel tools for TB vaccination and biomarker profiling. Our generic approach is applicable to other infectious diseases.We acknowledge funding by EC HORIZON2020 TBVAC2020 (Grant Agreement No. 643381); EC ITN FP7 VACTRAIN project (the text represents the authors’ views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information), The Netherlands Organization for Scientific Research (NWO-TOP Grant Agreement No. 91214038); European Research Council (ERC TB-ACCELERATE Grant Agreement No. 638553). Funding was also supplied by the Ministerio de Economía y Competitividad (Spanish Government) research grant SAF2013-43521-R, and the European Research Council (ERC) (638553-TB-ACCELERATE) (to IC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe