Identification of moaA3 gene in patient isolates of Mycobacterium tuberculosis in Kerala, which is absent in M. tuberculosis H37Rv and H37Ra

BACKGROUND: Tuberculosis is endemic to developing countries like India. Though the whole genome sequences of the type strain M. tuberculosis H37Rv and the clinical strain M. tuberculosis CDC1551 are available, the clinical isolates from India have not been studied extensively at the genome level. This study was carried out in order to have a better understanding of isolates from Kerala, a state in southern India. RESULTS: A PCR based strategy was followed making use of the deletion region primers to understand the genome level differences between the type strain H37Rv and the clinical isolates of M. tuberculosis from Kerala. PCR analysis of patient isolates using RD1 region primers revealed the amplification of a 386 bp region, in addition to the expected 652 bp amplicon. Southern hybridization of genomic DNA with the 386 bp amplicon confirmed the presence of this new region in a majority of the patient isolates from Kerala. Sequence comparison of this amplicon showed close homology with the moaA3 gene of M. bovis. In M. bovis this gene is present in the RvD5 region, an IS6110 mediated deletion that is absent in M. tuberculosis H37Rv. CONCLUSION: This study demonstrates the presence of moaA3 gene, that is absent in M. tuberculosis H37Rv and H37Ra, in a large number of local isolates. Whether the moaA3 gene provides any specific advantage to the field isolates of the pathogen is unclear. Field strains from Kerala have fewer IS6110 sequences and therefore are likely to have fewer IS6110 dependent rearrangements. But as deletions and insertions account for much of the genomic diversity of M. tuberculosis, the mechanisms of formation of sequence polymorphisms in the local isolates should be further examined. These results suggest that studies should focus on strains from endemic areas to understand the complexities of this pathogen

New tools for detecting latent tuberculosis infection: evaluation of RD1-specific long-term response

<p>Abstract</p> <p>Background</p> <p>Interferon-gamma (IFN-γ) release assays (IGRAs) were designed to detect latent tuberculosis infection (LTBI). However, discrepancies were found between the tuberculin skin test (TST) and IGRAs results that cannot be attributed to prior Bacille Calmètte Guerin vaccinations. The aim of this study was to evaluate tools for improving LTBI diagnosis by analyzing the IFN-γ response to RD1 proteins in prolonged (long-term response) whole blood tests in those subjects resulting negative to assays such as QuantiFERON-TB Gold In tube (QFT-IT).</p> <p>Methods</p> <p>The study population included 106 healthy TST⁺individuals with suspected LTBI (recent contact of smear-positive TB and homeless) consecutively enrolled. As controls, 13 healthy subjects unexposed to <it>M. tuberculosis </it>(TST^-, QFT-IT^-) and 29 subjects with cured pulmonary TB were enrolled. IFN-γ whole blood response to RD1 proteins and QFT-IT were evaluated at day 1 post-culture. A prolonged test evaluating long-term IFN-γ response (7-day) to RD1 proteins in diluted whole blood was performed.</p> <p>Results</p> <p>Among the enrolled TST⁺subjects with suspected LTBI, 70/106 (66.0%) responded to QFT-IT and 64/106 (60.3%) to RD1 proteins at day 1. To evaluate whether a prolonged test could improve the detection of LTBI, we set up the test using cured TB patients (with a microbiologically diagnosed past pulmonary disease) who resulted QFT-IT-negative and healthy controls as comparator groups. Using this assay, a statistically significant difference was found between IFN-γ levels in cured TB patients compared to healthy controls (p < 0.006). Based on these data, we constructed a receiver operating characteristic (ROC) curve and we calculated a cut-off. Based on the cut-off value, we found that among the 36 enrolled TST+ subjects with suspected LTBI not responding to QFT-IT, a long term response to RD1 proteins was detected in 11 subjects (30.6%).</p> <p>Conclusion</p> <p>These results indicate that IFN-γ long-term response to <it>M. tuberculosis </it>RD1 antigens may be used to detect past infection with <it>M. tuberculosis </it>and may help to identify additional individuals with LTBI who resulted negative in the short-term tests. These data may provide useful information for improving immunodiagnostic tests for tuberculosis infection, especially in individuals at high risk for active TB.</p

The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens

IP-10 response to RD1 antigens might be a useful biomarker for monitoring tuberculosis therapy

Background There is an urgent need of prognosis markers for tuberculosis (TB) to improve treatment strategies. The results of several studies show that the Interferon (IFN)-γ-specific response to the TB antigens of the QuantiFERON TB Gold (QFT-IT antigens) decreases after successful TB therapy. The objective of this study was to evaluate whether there are factors other than IFN-γ [such as IFN-γ inducible protein (IP)-10 which has also been associated with TB] in response to QFT-IT antigens that can be used as biomarkers for monitoring TB treatment. Methods In this exploratory study we assessed the changes in IP-10 secretion in response to QFT-IT antigens and RD1 peptides selected by computational analysis in 17 patients with active TB at the time of diagnosis and after 6 months of treatment. The IFN-γ response to QFT-IT antigens and RD1 selected peptides was evaluated as a control. A non-parametric Wilcoxon signed-rank test for paired comparisons was used to compare the continuous variables at the time of diagnosis and at therapy completion. A Chi-square test was used to compare proportions. Results We did not observe significant IP-10 changes in whole blood from either NIL or QFT-IT antigen tubes, after 1-day stimulation, between baseline and therapy completion (p = 0.08 and p = 0.7 respectively). Conversely, the level of IP-10 release to RD1 selected peptides was significantly different (p = 0.006). Similar results were obtained when we detected the IFN-γ in response to the QFT-IT antigens (p = 0.06) and RD1 selected peptides (p = 0.0003). The proportion of the IP-10 responders to the QFT-IT antigens did not significantly change between baseline and therapy completion (p = 0.6), whereas it significantly changed in response to RD1 selected peptides (p = 0.002). The proportion of IFN-γ responders between baseline and therapy completion was not significant for QFT-IT antigens (p = 0.2), whereas it was significant for the RD1 selected peptides (p = 0.002), confirming previous observations. Conclusions Our preliminary study provides an interesting hypothesis: IP-10 response to RD1 selected peptides (similar to IFN-γ) might be a useful biomarker for monitoring therapy efficacy in patients with active TB. However, further studies in larger cohorts are needed to confirm the consistency of these study results

Novel M tuberculosis Antigen-Specific T-Cells Are Early Markers of Infection and Disease Progression

Mycobacterium tuberculosis Region-of-Difference-1 gene products present opportunities for specific diagnosis of M. tuberculosis infection, yet immune responses to only two gene-products, Early Secretory Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10), have been comprehensively investigated.T-cell responses to Rv3873, Rv3878 and Rv3879c were quantified by IFN-γ-enzyme-linked-immunospot (ELISpot) in 846 children with recent household tuberculosis exposure and correlated with kinetics of tuberculin skin test (TST) and ESAT-6/CFP-10-ELISpot conversion over six months and clinical outcome over two years.Responses to Rv3873, Rv3878, and Rv3879c were present in 20-25% of contacts at enrolment. Rv3873 and Rv3879c responses were associated with and preceded TST conversion (P=0.02 and P=0.04 respectively), identifying these antigens as early targets of cell-mediated immunity following M. tuberculosis exposure. Responses to Rv3873 were additionally associated with subsequent ESAT-6/CFP-10-ELISpot conversion (P=0.04). Responses to Rv3873 and Rv3878 predicted progression to active disease (adjusted incidence rate ratio [95% CI] 3.06 [1.05,8.95; P=0.04], and 3.32 [1.14,9.71; P=0.03], respectively). Presence of a BCG-vaccination scar was associated with a 67% (P=0.03) relative risk reduction for progression to active tuberculosis.These RD1-derived antigens are early targets of cellular immunity following tuberculosis exposure and T-cells specific for these antigens predict progression to active tuberculosis suggesting diagnostic and prognostic utility

Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis

<div><h3>Background</h3><p>Depletion of T cells following infection by <em>Mycobacterium tuberculosis</em> (Mtb) impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised.</p> <h3>Methodology/Principal Findings</h3><p>We found that lymphopenia (<1.5×10⁹ cells/l) was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb) or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from <em>Mycobacterium bovis</em> Bacille de Calmette et Guerin (BCG)- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system.</p> <h3>Conclusions</h3><p>Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as interfere with microbial eradication in the granuloma.</p> </div

Strain-Specific Differences in the Genetic Control of Two Closely Related Mycobacteria

The host response to mycobacterial infection depends on host and pathogen genetic factors. Recent studies in human populations suggest a strain specific genetic control of tuberculosis. To test for mycobacterial-strain specific genetic control of susceptibility to infection under highly controlled experimental conditions, we performed a comparative genetic analysis using the A/J- and C57BL/6J-derived recombinant congenic (RC) mouse panel infected with the Russia and Pasteur strains of Mycobacterium bovis Bacille Calmette Guérin (BCG). Bacillary counts in the lung and spleen at weeks 1 and 6 post infection were used as a measure of susceptibility. By performing genome-wide linkage analyses of loci that impact on tissue-specific bacillary burden, we were able to show the importance of correcting for strain background effects in the RC panel. When linkage analysis was adjusted on strain background, we detected a single locus on chromosome 11 that impacted on pulmonary counts of BCG Russia but not Pasteur. The same locus also controlled the splenic counts of BCG Russia but not Pasteur. By contrast, a locus on chromosome 1 which was indistinguishable from Nramp1 impacted on splenic bacillary counts of both BCG Russia and Pasteur. Additionally, dependent upon BCG strain, tissue and time post infection, we detected 9 distinct loci associated with bacillary counts. Hence, the ensemble of genetic loci impacting on BCG infection revealed a highly dynamic picture of genetic control that reflected both the course of infection and the infecting strain. This high degree of adaptation of host genetics to strain-specific pathogenesis is expected to provide a suitable framework for the selection of specific host-mycobacteria combinations during co-evolution of mycobacteria with humans

Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Studies of <it>Mycobacterium bovis </it>BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico.</p> <p>Results</p> <p>BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests.</p> <p>Conclusions</p> <p>BCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (<it>fadD23</it>) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time.</p

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective

LTC: a novel algorithm to improve the efficiency of contig assembly for physical mapping in complex genomes

<p>Abstract</p> <p>Background</p> <p>Physical maps are the substrate of genome sequencing and map-based cloning and their construction relies on the accurate assembly of BAC clones into large contigs that are then anchored to genetic maps with molecular markers. High Information Content Fingerprinting has become the method of choice for large and repetitive genomes such as those of maize, barley, and wheat. However, the high level of repeated DNA present in these genomes requires the application of very stringent criteria to ensure a reliable assembly with the FingerPrinted Contig (FPC) software, which often results in short contig lengths (of 3-5 clones before merging) as well as an unreliable assembly in some difficult regions. Difficulties can originate from a non-linear topological structure of clone overlaps, low power of clone ordering algorithms, and the absence of tools to identify sources of gaps in Minimal Tiling Paths (MTPs).</p> <p>Results</p> <p>To address these problems, we propose a novel approach that: (i) reduces the rate of false connections and Q-clones by using a new cutoff calculation method; (ii) obtains reliable clusters robust to the exclusion of single clone or clone overlap; (iii) explores the topological contig structure by considering contigs as networks of clones connected by significant overlaps; (iv) performs iterative clone clustering combined with ordering and order verification using re-sampling methods; and (v) uses global optimization methods for clone ordering and Band Map construction. The elements of this new analytical framework called Linear Topological Contig (LTC) were applied on datasets used previously for the construction of the physical map of wheat chromosome 3B with FPC. The performance of LTC vs. FPC was compared also on the simulated BAC libraries based on the known genome sequences for chromosome 1 of rice and chromosome 1 of maize.</p> <p>Conclusions</p> <p>The results show that compared to other methods, LTC enables the construction of highly reliable and longer contigs (5-12 clones before merging), the detection of "weak" connections in contigs and their "repair", and the elongation of contigs obtained by other assembly methods.</p