Project description:In this study we have combined RNA-seq analysis of genome-wide transcriptional start sites with regular RNA-seq to study the transcriptional landscape of Mycobacterium tuberculosis during exponential culture and growth arrest using a starvation model where exponentially growing cells are incubated in PBS for 24 hours. Three independent biological replicates were used in this experiment.
Project description:Mycobacterium tuberculosis is an intracellular human pathogen with the ability to resist and adapt to many adverse conditions it encounters upon infection. Among these, overcoming the production of nitric oxide by macrophages could be key for M. tuberculosis success. We have challenged M. tuberculosis with a sub-lethal concentration of nitric oxide and followed the transcriptomic response through RNA-seq for 48 hours.
Project description:This study was aimed towards understanding the consequences of mutating rv2887, a putative transcriptional repressor, on gene expression in Mycobacterium tuberculosis. Since clones carrying mutations in this gene were found to be highly resistant to a potent mycobactericidal compound, 14, we also tested the impact of short-term exposure to this compound on gene expression in Mycobacterium tuberculosis. Three different strains, wild type H37Rv and the two clones resistant to compound 14, named 1A and 8A, were grown to log phase in 7H9 complete medium. The cells were diluted to an OD of 0.5 and split in to two equal parts, 1 arm received the vehicle (DMSO) and the other arm received compound 14 at a final concentration of 3.9 μM. The exposure lasted 4 hours, at which point the bacterial pellet was collected and processed for RNA extraction using protocols described previously.
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Our prediction suggested that Mcr7 might regulate tatC at the post-transcriptional level by occlusion of the RBS and the consequent translational arrest. Consequently, we studied the secretome from exponentially grown cultures of strain H37Rv, its phoP mutant and a phoP complemented mutant by in-depth proteomics. The results are consistent with a regulatory model involving PhoP, Mcr7 and tatC mRNA since the absence of Mcr7 in the phoP mutant would result in more efficient TatC translation and therefore increased secretion.
Project description:Transcriptional changes during early infection of macrophage-like THP-1 cell line with pathogenic bacterium Mycobacterium tuberculosis. RNAseq samples were taken at 0h (THP-1 cells growing in the RPMI medium), and after 4h, 24h and 48h post infection. Bacterial enrichment was performed to increase the amount of bacterial mRNA in the samples. Non-enriched samples were used to map THP-1 cells transcripts; enriched samples were used to map M. tuberculosis transcripts the corresponding genomes.
Project description:The global diversity of Mycobacterium tuberculosis comprises at least seven lineages, each with its distinct geographic distribution. The aim of this experiment was to perform a comparative analysis of two of these lineages: Lineage 1 and Lineage 2. The former is found around the rim of the Indian ocean and in south-east Asia, while the latter is widely spread throughout Asia and shows an increasing global spread. We have chosen three fully drug susceptible clincal isolates to represent each of the two lineages. We performed RNAseq analysis on rRNA depleted samples isolated from cultures during mid-log phase. Each strain was measured in triplicate.
Project description:The experiment was designed to infer the fitness cost of rifampicin-resistance in Mycobacterium tuberculosis through expression analysis. The approach relied on: 1. Tracking the expression changes occurring as a result of the rifampicin-resistance conferring mutation Ser450Leu in RpoB and subsequent gain of compensatory mutation Leu516Pro in RpoC. The hypothesis was that any cost-incurring expressional changes would be reversed in the presence of compensatory mutations. The strains in this set were described before here: PMID 22179134. 2. Comparing the impact of the same rifampicin-resistance mutation (RpoB Ser450Leu) in five different genetic backgrounds. Here the comparison was solely between RpoB Ser450Leu and their cognate drug susceptible wild type ancestor. One of the strain pairs is the same as in point 1. above.
Project description:We analyzed the genes expressed, or the transcriptome, of bacilli (Mycobacterium tuberculosis) growing in fatty acids as sole carbon source. Using new technologies to massively sequence of RNA molecules we identified a group of genes that provides novel insight regarding the metabolic pathways and transcriptional regulation of latent M. Tuberculosis. Comparative Transcriptomics between two carbon source (Dextrose, Long Fatty Acids), at two states of growth (Exponential and Stationary Phase)
Project description:Tuberculosis (TB) is still a major global health challenge, killing over 1.5 million people each year, and hence, there is a need to identify and develop novel treatments for Mycobacterium tuberculosis (M. tuberculosis). The prevalence of infections caused by nontuberculous mycobacteria (NTM) is also increasing and has overtaken TB cases in the United States and much of the developed world. Mycobacterium abscessus (M. abscessus) is one of the most frequently encountered NTM and is difficult to treat. We describe the use of drug-disease association using a semantic knowledge graph approach combined with machine learning models that has enabled the identification of several molecules for testing anti-mycobacterial activity. We established that niclosamide (M. tuberculosis IC90 2.95 μM; M. abscessus IC90 59.1 μM) and tribromsalan (M. tuberculosis IC90 76.92 μM; M. abscessus IC90 147.4 μM) inhibit M. tuberculosis and M. abscessus in vitro. To investigate the mode of action, we determined the transcriptional response of M. tuberculosis and M. abscessus to both compounds in axenic log phase, demonstrating a broad effect on gene expression that differed from known M. tuberculosis inhibitors. Both compounds elicited transcriptional responses indicative of respiratory pathway stress and the dysregulation of fatty acid metabolism. Further testing against drug-resistant isolates and other NTM is warranted to clarify the usefulness of these repurposed drugs for mycobacteria.
Project description:The Mycobacterium tuberculosis eukaryotic-like serine/threonine protein kinase (STPKs) PknA and PknB regulate several processes required for cell growth, both kinase have been found to be and can be investigated by using the conditional mutant. In the present study, we created the M. tuberculosis kinase PknA/ PknB depletion mutant and performed the phosphoproteomics to uncover phosphorylation substrates changes when PknA/ PknB depletion.