Transcriptional Response of Mycobacterium tuberculosis H37Rv to BTZ043
ABSTRACT: To understand the response of M. tuberculosis (MTB) to the drug BTZ043, we performed transcriptomics on MTB bacilli exposed to the drug. Overall design: Bacteria were harvested at 0,1 day after being exposed to concentrations of BTZ043 (no drug, 0.006μM) as indicated in each sample title. RNA was isolated and gene expression quantified by sequencing.
Project description:To understand the response of M. tuberculosis (MTB) to the first-line drug P218, we performed transcriptomics on MTB bacilli exposed to the drug. Overall design: Bacteria were harvested at 0,1 day after being exposed to concentrations of P218 (no drug, 0.1μM) as indicated in each sample title. RNA was isolated and gene expression quantified by sequencing.
Project description:To understand the response of M. tuberculosis (MTB) to the first-line drug clarithromycin, we performed transcriptomics on MTB bacilli exposed to the drug. Overall design: Bacteria were harvested at 0,1 day after being exposed to concentrations of clarithromycin (no drug, 20μM) as indicated in each sample title. RNA was isolated and gene expression quantified by sequencing.
Project description:Bacteria commonly adapt to stresses by altering gene expression. To understand the response of M. tuberculosis (MTB) to various antibacterial agents, we performed transcriptomics on MTB bacilli exposed to several test compounds as well as known drugs (capreomycin, cycloserine, ethionamide, isoniazid, kanamycin, moxifloxacin, PA-824, rifampicin, streptomycin). Bacteria were exposed for 16 hrs to various concentrations of each drug (different multiples of the compound's MIC), as noted in the title of each sample. RNA was isolated and applied to arrays provided by TIGR under the NIAID contract N01-AI-15447
Project description:Bacteria commonly adapt to stresses by altering gene expression. To understand the response of M. tuberculosis (MTB) to bedaquiline, we performed transcriptomics over a time-course on MTB bacilli exposed to the drug. Overall design: Bacteria were harvested at 1, 6, 24, 48, 96 hrs after being exposed to concentrations of bedaquiline (no drug, 1.5μM, 15μM) during a 7-day drug exposure time course experiment (as indicated in each sample title). RNA was isolated and applied to custom Agilent tiled arrays. Please note that experiments were performed technically as dual channel (eg, Cy3 and Cy5-labeled samples hybridized to the same array) but processed the results as though they are single channel (Cy3 and Cy5 signals are calculated). Therefore, there are two sample records per each raw data file and the raw data file associated with each sample is indicated in the corresponding sample description field.
Project description:BACKGROUND:Mycobacterium tuberculosis (MTB) is a common bacterium causing tuberculosis and remains a major pathogen for mortality. Although the MTB genome has been extensively explored for two decades, the functions of 27% (1051/3906) of encoded proteins have yet to be determined and these proteins are annotated as hypothetical proteins. METHODS:We assigned functions to these hypothetical proteins using SSEalign, a newly designed algorithm utilizing structural information. A set of rigorous criteria was applied to these annotations in order to examine whether they were supported by each parameter. Virulence factors and potential drug targets were also screened among the annotated proteins. RESULTS:For 78% (823/1051) of the hypothetical proteins, we could identify homologs in Escherichia coli and Salmonella typhimurium by using SSEalign. Functional classification analysis indicated that 62.2% (512/823) of these annotated proteins were enzymes with catalytic activities and most of these annotations were supported by at least two other independent parameters. A relatively high proportion of transporter was identified in MTB genome, indicating the potential frequent transportation of frequent absorbing essential metabolites and excreting toxic materials in MTB. Twelve virulence factors and ten vaccine candidates were identified within these MTB hypothetical proteins, including two genes (rpoS and pspA) related to stress response to the host immune system. Furthermore, we have identified six novel drug target candidates among our annotated proteins, including Rv0817 and Rv2927c, which could be used for treating MTB infection. CONCLUSIONS:Our annotation of the MTB hypothetical proteins will probably serve as a useful dataset for future MTB studies.
Project description:Despite the widespread use of the childhood vaccine against tuberculosis (TB), Mycobacterium bovis bacillus Calmette-Guérin (BCG), the disease remains a serious global health problem. A successful vaccine against TB that replaces or boosts BCG would include antigens that induce or recall the appropriate T cell responses. Four Mycobacterium tuberculosis (Mtb) antigens--including members of the virulence factor families PE/PPE and EsX or antigens associated with latency--were produced as a single recombinant fusion protein (ID93). When administered together with the adjuvant GLA-SE, a stable oil-in-water nanoemulsion, the fusion protein was immunogenic in mice, guinea pigs, and cynomolgus monkeys. In mice, this fusion protein-adjuvant combination induced polyfunctional CD4 T helper 1 cell responses characterized by antigen-specific interferon-?, tumor necrosis factor, and interleukin-2, as well as a reduction in the number of bacteria in the lungs of animals after they were subsequently infected with virulent or multidrug-resistant Mtb strains. Furthermore, boosting BCG-vaccinated guinea pigs with fusion peptide-adjuvant resulted in reduced pathology and fewer bacilli, and prevented the death of animals challenged with virulent Mtb. Finally, the fusion protein elicited polyfunctional effector CD4 and CD8 T cell responses in BCG-vaccinated or Mtb-exposed human peripheral blood mononuclear cells. This study establishes that the protein subunit vaccine consisting of the fusion protein and adjuvant protects against TB and drug-resistant TB in animals and is a candidate for boosting the protective efficacy of the childhood BCG vaccine in humans.
Project description:Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25 A resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism.
Project description:Mycobacterium tuberculosis (Mtb) forms biofilms harbouring antibiotic-tolerant bacilli in vitro, but the factors that induce biofilm formation and the nature of the extracellular material that holds the cells together are poorly understood. Here we show that intracellular thiol reductive stress (TRS) induces formation of Mtb biofilms in vitro, which harbour drug-tolerant but metabolically active bacteria with unchanged levels of ATP/ADP, NAD(+)/NADH and NADP(+)/NADPH. The development of these biofilms requires DNA, RNA and protein synthesis. Transcriptional analysis suggests that Mtb modulates only ?7% of its genes for survival in biofilms. In addition to proteins, lipids and DNA, the extracellular material in these biofilms is primarily composed of polysaccharides, with cellulose being a key component. Our results contribute to a better understanding of the mechanisms underlying Mtb biofilm formation, although the clinical relevance of Mtb biofilms in human tuberculosis remains unclear.
Project description:Mycobacterium tuberculosis (Mtb) infection results in a spectrum of clinical and histopathologic manifestations. It has been proposed that the environmental and immune pressures associated with different contexts of infection have different consequences for the associated bacterial populations, affecting drug susceptibility and the emergence of resistance. However, there is little concrete evidence for this model. We prospectively collected sputum samples from 18 newly diagnosed and treatment-naïve patients with tuberculosis and sequenced 795 colony-derived Mtb isolates. Mutant accumulation rates varied considerably between different bacilli isolated from the same individual, and where high rates of mutation were observed, the mutational spectrum was consistent with reactive oxygen species-induced mutagenesis. Elevated bacterial mutation rates were identified in isolates from HIV-negative but not HIV-positive individuals, suggesting that they were immune-driven. These results support the model that mutagenesis of Mtb in vivo is modulated by the host environment, which could drive the emergence of variants associated with drug resistance in a host-dependent manner.
Project description:BACKGROUND: Mycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis, killing ~1.7 million people annually. The remarkable capacity of this pathogen to escape the host immune system for decades and then to cause active tuberculosis disease, makes M.tb a successful pathogen. Currently available anti-mycobacterial therapy has poor compliance due to requirement of prolonged treatment resulting in accelerated emergence of drug resistant strains. Hence, there is an urgent need to identify new chemical entities with novel mechanism of action and potent activity against the drug resistant strains. RESULTS: This study describes novel computational models developed for predicting inhibitors against both replicative and non-replicative phase of drug-tolerant M.tb under carbon starvation stage. These models were trained on highly diverse dataset of 2135 compounds using four classes of binary fingerprint namely PubChem, MACCS, EState, SubStructure. We achieved the best performance Matthews correlation coefficient (MCC) of 0.45 using the model based on MACCS fingerprints for replicative phase inhibitor dataset. In case of non-replicative phase, Hybrid model based on PubChem, MACCS, EState, SubStructure fingerprints performed better with maximum MCC value of 0.28. In this study, we have shown that molecular weight, polar surface area and rotatable bond count of inhibitors (replicating and non-replicating phase) are significantly different from non-inhibitors. The fragment analysis suggests that substructures like hetero_N_nonbasic, heterocyclic, carboxylic_ester, and hetero_N_basic_no_H are predominant in replicating phase inhibitors while hetero_O, ketone, secondary_mixed_amine are preferred in the non-replicative phase inhibitors. It was observed that nitro, alkyne, and enamine are important for the molecules inhibiting bacilli residing in both the phases. In this study, we introduced a new algorithm based on Matthews correlation coefficient called MCCA for feature selection and found that this algorithm is better or comparable to frequency based approach. CONCLUSION: In this study, we have developed computational models to predict phase specific inhibitors against drug resistant strains of M.tb grown under carbon starvation. Based on simple molecular properties, we have derived some rules, which would be useful in robust identification of tuberculosis inhibitors. Based on these observations, we have developed a webserver for predicting inhibitors against drug tolerant M.tb H37Rv available at http://crdd.osdd.net/oscadd/mdri/.