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: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:Bacterial pathogens exploit secreted aminopeptidases to modulate host cellular functions. Mycobacterium tuberculosis (Mtb) secretes PepN, a M1 aminopeptidase with N-terminal peptidase and C-terminal ERAP1_C-like domains. In addition to being conserved across Mtb complex, PepNMtb is also homologous (78% amino acids identity) to PepN of M. smegmatis (Msmeg), a non-pathogenic mycobacterium. Despite high homogeneity, PepNMtb and PepNMsmeg exhibit opposing traits thus providing insights into their plausible bacterial/host-specific functions. Our biochemical studies show that, while both PepNs uniformly accumulate across all stages of in vitro growth, unlike Mtb, Msmeg is intolerant to over accumulation of its PepN and hence robustly proteolyzes it. Our proteomic analyses indicate Msmeg also secreting some of its excess PepN into its culture supernatant. In contrast, Mtb does not excessively secrete or proteolyse its over accumulating PepNMtb. Our fractionation studies show Mtb translocating its full length PepNMtb into its membrane and cell wall fractions. Instead, Msmeg accumulates its full length PepNMsmeg in its cytosol, and promotes its cleaved (~40 kDa) form to locate into subcellular fractions. PepNMtb is redundant for Mtb’s in vitro growth and thus Mtb is readily amenable to ΔpepN generation. Opposingly, PepNMsmeg is necessary but not sufficient for Msmeg growth in vitro. Given such disparity, co-immunoprecipitation studies on both PepNs predictably showed divergent interactomes with minimal common hits. In our in vitro infection studies, PepNMtb localizes host endoplasmic reticulum directing us to its potential host-specific role. In summary, our study provides insights into PepNs dichotomy despite them being conserved across pathogenic and non-pathogenic mycobacterial species.
Project description:Mycobacterium tuberculosis, the causative agent of tuberculosis accounts for 1.5 million annual deaths worldwide. The two well-characterized strains of the parental H37 strain namely, H37Ra and H37Rv show different pathogenic phenotypes. In order to identify factors that are responsible for virulence, we compared the proteome and the phosphoproteome profiles of virulent (H37Rv) and virulence attenuated (H37Ra) strains of M. tuberculosis. Quantitative proteomic analysis resulted in the identification and quantitation of 2,709 proteins and 505 phosphosites. Comparative analysis revealed over 5-fold overexpression of several proteins associated with virulence. Our data indicates that there are definable molecular differences between H37Rv and H37Ra strains at both the proteome and phosphoproteome levels which may explain the virulence and phenotypic differences.
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: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 a facultative intracellular pathogen, responsible for causing tuberculosis. The harsh environment in which M. tuberculosis survives requires this pathogen to maintain an evolutionary advantage. However, the apparent absence of horizontal gene transfer in M. tuberculosis imposes restrictions in the ways by which evolution can occur. Large scale changes in the genome can be introduced through genome reduction, recombination events and structural variation. Here, we identify a functional chimeric protein in the ppe38-71 locus, the absence of which is known to have an impact on protein secretion and virulence. To examine whether this approach was used more often by this pathogen we further develop software that detects potential gene fusion events from multigene deletions using whole-genome sequencing data. With this software we could identify a number of other putative gene-fusion events within the genomes of M. tuberculosis isolates. We were able to demonstrate the expression of one of these gene fusions at the protein level using mass spectrometry. Therefore, gene fusions may provide an additional means of evolution for M. tuberculosis in its natural environment whereby novel proteins and functions can arise.
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.