Improving isothermal DNA amplification speed for the rapid detection of Mycobacterium tuberculosis

In this study, we report the development of a helicase-dependent amplification assay for rapid detection of Mycobacterium tuberculosis (MTB). By applying a step-by-step optimization method, the amplification time from an input of 2-copy MTB genomic DNA was reduced from about 60 min to less than 30 min.     

Construction of a virtual Mycobacterium tuberculosis consensus genome and its application to data from a next generation sequencer

Background

Although Mycobacterium tuberculosis isolates are consisted of several different lineages and the epidemiology analyses are usually assessed relative to a particular reference genome, M. tuberculosis H37Rv, which might introduce some biased results. Those analyses are essentially based genome sequence information of M. tuberculosis and could be performed in sillico in theory, with whole genome sequence (WGS) data available in the databases and obtained by next generation sequencers (NGSs). As an approach to establish higher resolution methods for such analyses, whole genome sequences of the M. tuberculosis complexes (MTBCs) strains available on databases were aligned to construct virtual reference genome sequences called the consensus sequence (CS), and evaluated its feasibility in in sillico epidemiological analyses.

Results

The consensus sequence (CS) was successfully constructed and utilized to perform phylogenetic analysis, evaluation of read mapping efficacy, which is crucial for detecting single nucleotide polymorphisms (SNPs), and various MTBC typing methods virtually including spoligotyping, VNTR, Long sequence polymorphism and Beijing typing. SNPs detected based on CS, in comparison with H37Rv, were utilized in concatemer-based phylogenetic analysis to determine their reliability relative to a phylogenetic tree based on whole genome alignment as the gold standard. Statistical comparison of phylogenic trees based on CS with that of H37Rv indicated the former showed always better results that that of later. SNP detection and concatenation with CS was advantageous because the frequency of crucial SNPs distinguishing among strain lineages was higher than those of H37Rv. The number of SNPs detected was lower with the consensus than with the H37Rv sequence, resulting in a significant reduction in computational time. Performance of each virtual typing was satisfactory and accorded with those published when those are available.

Conclusions

These results indicated that virtual CS constructed from genome sequence data is an ideal approach as a reference for MTBC studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1368-9) contains supplementary material, which is available to authorized users.     

Synthetic Biology in Action: Developing a Drug Against MDR-TB

The amalgamation of the research efforts of biologists, chemists and geneticists led by scientists at the Department of Zoology, University of Delhi has resulted in the development of a novel rifamycin derivative; 24-desmethylrifampicin, which is highly effective against multi-drug resistant (MDR) strains of Mycobacterium tuberculosis. The production of rifamycin analogue was facilitated by genetic-synthetic strategies that have opened an interdisciplinary route for the development of more such rifamycin analogues aiming at a better therapeutic potential. The results of this painstaking effort of nearly 25 years of a team of students and scientists led by Professor Rup Lal have been recently published in the Journal of Biological Chemistry (www.jbc.org/content/289/30/21142.long). This strategy can now find applications for developing newer rifamycin analogues that can be harnessed to overcome the problem of MDR, extensively drug resistant (XDR) and totally drug resistant (TDR) M. tuberculosis.     

Mycobacterial induction of autophagy varies by species and occurs independently of mammalian target of rapamycin inhibition

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to latent infection. Although many factors are involved, the mammalian autophagy pathway is recognized as a determinant that can influence the course of infection. Intervention aimed at utilizing autophagy to clear infection requires an examination of the autophagy and signal transduction induced by mycobacteria under native conditions. With both pathogenic and non-pathogenic mycobacteria, we show that infection correlates with an increase in the mammalian target of rapamycin (mTOR) activity indicating that autophagy induction by mycobacteria occurs in an mTOR-independent manner. Analysis of Mycobacterium smegmatis and Mycobacterium bovis bacille Calmette-Guérin (BCG), which respectively induce high and low autophagy responses, indicates that lipid material is capable of inducing both autophagy and mTOR signaling. Although mycobacterial infection potently induces mTOR activity, we confirm that bacterial viability can be reduced by rapamycin treatment. In addition, our work demonstrates that BCG can reduce autophagy responses to M. smegmatis suggesting that specific mechanisms are used by BCG to minimize host cell autophagy. We conclude that autophagy induction and mTOR signaling take place concurrently during mycobacterial infection and that host autophagy responses to any given mycobacterium stem from multiple factors, including the presence of activating macromolecules and inhibitory mechanisms.     

Resistome analysis of Mycobacterium tuberculosis: Identification of aminoglycoside 2'-Nacetyltransferase (AAC) as co-target for drug desigining

The emergence of multidrug resistant tuberculosis (MDRTB) highlights the urgent need to understand the mechanisms of resistance to the drugs and to develop a new arena of therapeutics to treat the disease. Ethambutol, isonazid, pyrazinamide, rifampicin are first line of drugs against TB, whereas aminoglycoside, polypeptides, fluoroquinolone, ethionamide are important second line of bactericidal drugs used to treat MDRTB, and resistance to one or both of these drugs are defining characteristic of extensively drug resistant TB. We retrieved 1,221 resistant genes from Antibiotic Resistance Gene Database (ARDB), which are responsible for resistance against first and second line antibiotics used in treatment of Mycobacterium tuberculosis infection. From network analysis of these resistance genes, 53 genes were found to be common. Phylogenetic analysis shows that more than 60% of these genes code for acetyltransferase. Acetyltransferases detoxify antibiotics by acetylation, this mechanism plays central role in antibiotic resistance. Seven acetyltransferase (AT-1 to AT-7) were selected from phylogenetic analysis. Structural alignment shows that these acetyltransferases share common ancestral core, which can be used as a template for structure based drug designing. From STRING analysis it is found that acetyltransferase interact with 10 different proteins and it shows that, all these interaction were specific to M. tuberculosis. These results have important implications in designing new therapeutic strategies with acetyltransferase as lead co-target to combat against MDR as well as Extreme drug resistant (XDR) tuberculosis.

Abbreviations

AA - amino acid, AT - Acetyltransferase, AAC - Aminoglycoside 2''-N-acetyltransferase, XDR - Extreme drug-resistant, MDR - Multidrug-resistant, Mtb - Mycobacterium tuberculosis, TB - Tuberculosis.     

The ongoing challenge of latent tuberculosis

The global health community has set itself the task of eliminating tuberculosis (TB) as a public health problem by 2050. Although progress has been made in global TB control, the current decline in incidence of 2% yr⁻¹ is far from the rate needed to achieve this. If we are to succeed in this endeavour, new strategies to reduce the reservoir of latently infected persons (from which new cases arise) would be advantageous. However, ascertainment of the extent and risk posed by this group is poor. The current diagnostics tests (tuberculin skin test and interferon-gamma release assays) poorly predict who will develop active disease and the therapeutic options available are not optimal for the scale of the intervention that may be required. In this article, we outline a basis for our current understanding of latent TB and highlight areas where innovation leading to development of novel diagnostic tests, drug regimens and vaccines may assist progress. We argue that the pool of individuals at high risk of progression may be significantly smaller than the 2.33 billion thought to be immune sensitized by Mycobacterium tuberculosis and that identifying and targeting this group will be an important strategy in the road to elimination.     

Using a Label Free Quantitative Proteomics Approach to Identify Changes in Protein Abundance in Multidrug-Resistant Mycobacterium tuberculosis

Reports in recent years indicate that the increasing emergence of resistance to drugs be using to TB treatment. The resistance to them severely affects to options for effective treatment. The emergence of multidrug-resistant tuberculosis has increased interest in understanding the mechanism of drug resistance in M. tuberculosis and the development of new therapeutics, diagnostics and vaccines. In this study, a label-free quantitative proteomics approach has been used to analyze proteome of multidrug-resistant and susceptible clinical isolates of M. tuberculosis and identify differences in protein abundance between the two groups. With this approach, we were able to identify a total of 1,583 proteins. The majority of identified proteins have predicted roles in lipid metabolism, intermediary metabolism, cell wall and cell processes. Comparative analysis revealed that 68 proteins identified by at least two peptides showed significant differences of at least twofolds in relative abundance between two groups. In all protein differences, the increase of some considering proteins such as NADH dehydrogenase, probable aldehyde dehydrogenase, cyclopropane mycolic acid synthase 3, probable arabinosyltransferase A, putative lipoprotein, uncharacterized oxidoreductase and six membrane proteins in resistant isolates might be involved in the drug resistance and to be potential diagnostic protein targets. The decrease in abundance of proteins related to secretion system and immunogenicity (ESAT-6-like proteins, ESX-1 secretion system associated proteins, O-antigen export system and MPT63) in the multidrug-resistant strains can be a defensive mechanism undertaken by the resistant cell.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0511-2) contains supplementary material, which is available to authorized users.     

Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.     

Sublineages of Beijing Strain of Mycobacterium tuberculosis in Sri Lanka

Strains of the Beijing/W genotype of Mycobacterium tuberculosis have been responsible for large outbreaks of tuberculosis around the world, sometimes involving multi-drug resistance. It has been shown that more recently evolved Beijing sublineages are prone to cause outbreaks. Furthermore Beijing is the single predominant cluster in Sri Lanka. The present study identifies that recently evolved sublineages of Beijing strains are present in the study population. The majority of Beijing isolates (92.85%) were pan-susceptible. However, these findings may have important implications for the control and prevention of tuberculosis in Sri Lanka.     

Recombinant antigen production for assays of intradermoreaction for diagnosis and surveillance of tuberculosis

The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway^® system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value = 0.00386).     

The Tussle Between Mycobacteria and Host: To Eat or Not To Eat

Autophagy is a catabolic process of cellular homeostasis evolutionarily conserved in eukaryotes. To block infection of intracellular bacterial pathogens, metazoans deploy autophagy for pathogen clearance through phago-lysosome formation and specific bactericidal peptides. Although an array of research have publicized the host regulatory factors, the function of bacterial effectors are yet to be understood in detail. In this article, we focus on the autophagic response to one of the most successful intracellular bacteria Mycobacterium tuberculosis.     

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.     

Insights from the docking and molecular dynamics simulation of the Phosphopantetheinyl transferase (PptT) structural model from Mycobacterium tuberculosis

A great challenge is posed to the treatment of tuberculosis due to the evolution of multidrug-resistant (MDR) and extensively drugresistant (XDR) strains of Mycobacterium tuberculosis in recent times. The complex cell envelope of the bacterium contains unusual structures of lipids which protects the bacterium from host enzymes and escape immune response. To overcome the drug resistance, targeting “drug targets” which have a critical role in growth and virulence factor is a novel approach for better tuberculosis treatment. The enzyme Phosphopantetheinyl transferase (PptT) is an attractive drug target as it is primarily involved in post translational modification of various types-I polyketide synthases and assembly of mycobactin, which is required for lipid virulence factors. Our in silico studies reported that the structural model of M.tuberculosis PptT characterizes the structure-function activity. The refinement of the model was carried out with molecular dynamics simulations and was analyzed with root mean square deviation (RMSD), and radius of gyration (Rg). This confirmed the structural behavior of PptT in dynamic system. Molecular docking with substrate coenzyme A (CoA) identified the binding pocket and key residues His93, Asp114 and Arg169 involved in PptT-CoA binding. In conclusion, our results show that the M.tuberculosis PptT model and critical CoA binding pocket initiate the inhibitor design of PptT towards tuberculosis treatment.     

Thiamin (Vitamin B1) Biosynthesis and Regulation: A Rich Source of Antimicrobial Drug Targets?

Drug resistance of pathogens has necessitated the identification of novel targets for antibiotics. Thiamin (vitamin B1) is an essential cofactor for all organisms in its active form thiamin diphosphate (ThDP). Therefore, its metabolic pathways might be one largely untapped source of antibiotics targets. This review describes bacterial thiamin biosynthetic, salvage, and transport pathways. Essential thiamin synthetic enzymes such as Dxs and ThiE are proposed as promising drug targets. The regulation mechanism of thiamin biosynthesis by ThDP riboswitch is also discussed. As drug targets of existing antimicrobial compound pyrithiamin, the ThDP riboswitch might serves as alternative targets for more antibiotics.     

Identifying control mechanisms of granuloma formation during M. tuberculosis infection using an agent-based model

Infection with Mycobacterium tuberculosis is a major world health problem. An estimated 2 billion people are presently infected and the disease causes approximately 3 million deaths per year. After bacteria are inhaled into the lung, a complex immune response is triggered leading to the formation of multicellular structures termed granulomas. It is believed that the collection of host granulomas either contain bacteria resulting in a latent infection or are unable to do so, leading to active disease. Thus, understanding granuloma formation and function is essential for improving both diagnosis and treatment of tuberculosis. Granuloma formation is a complex spatio-temporal system involving interactions of bacteria, specific immune cells, including macrophages, CD4+ and CD8+ T cells, as well as immune effectors such as chemokine and cytokines. To study this complex dynamical system we have developed an agent-based model of granuloma formation in the lung. This model combines continuous representations of chemokines with discrete agent representations of macrophages and T cells in a cellular automata-like environment. Our results indicate that key host elements involved in granuloma formation are chemokine diffusion, prevention of macrophage overcrowding within the granuloma, arrival time, location and number of T cells within the granuloma, and an overall host ability to activate macrophages. Interestingly, a key bacterial factor is its intracellular growth rate, whereby slow growth actually facilitates survival.