Project description:Feature reduction of microarray data from mycobacteria treated with a variety of various clinical and investigational drugs We are using feature reduction to demonstrate that subsets of biomarker genes representative of the whole genome are sufficient for MOA classification and deconvolution in a medium-throughput microfluidic format ultimately leading to a cost effective and rapid tool for routine antibacterial drug-discovery programs.
Project description:Mycobacteria are known to be non-spore forming but very hardy: the bacilli can for instance survive starvation in zero-nutrient saline in a non-replicating state. Recently we reported that mycobacteria in fact can undergo cellular differentiation when exposed to different starvation conditions. The presence of traces of nutrients triggers the development of a new, ‘small resting cell’ form (SMRCs). Saline shock-starved large resting cells (LARCs), which did not show cell size or surface changes when observed by scanning electron microscopy, remodeled their internal structure to the septated, multi-nucleoided cells seen during differentiation to SMRCs. Here we conduct RNA-seq to gain greater insights into whether starvation elicited a distinct developmental pathway. Comparative transcriptome analysis of SMRC and LARC development revealed largely overlapping sets of differentially expressed regulatory and metabolic genes. These transcriptome data are consistent with a mycobacterial starvation-induced differentiation program in which at first septated, multi-nuceloided cells are generated. Under zero-nutrient conditions bacteria terminate development at this stage as LARCs. In the presence of traces of a carbon source, these multi-nucleoided cells continue differentiation into mono-nuleoided SMRCs.
Project description:CarD is essential transcription factors in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strains with CarD-FLAG (LK2539), where an additional copy of CarD under anhydrotetracycline inducible promoter was stably inserted into the genome. CarD-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth.
Project description:RbpA is an essential transcription factors in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strains with RbpA-FLAG (LK2541), where an additional copy of RbpA under anhydrotetracycline inducible promoter was stably inserted into the genome. RbpA-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth.
Project description:CsrL (MSMEG_5890) associates with CarD and RNA polymerase in mycobacteria. For ChIP-seq, we used Mycobacterium smegmatis strain with CrsL (LK3051), where an additional copy of CrsL under anhydrotetracycline inducible promoter was stably inserted into M. smegmatis genome. CrsL-FLAG was expressed in exponential phase and ChIP performed at mid-exponential phase of growth.
Project description:The cytokine interferon-γ is a principal effector of macrophage activation and immune resistance to mycobacterial infection; however, pathogenic mycobacteria are capable of surviving in interferon-γ-activated macrophages by largely unknown mechanisms. We found that interferon-γ specifically bound to pathogenic mycobacteria and enhanced their growth in culture. Proteomic and electron microscopy analyses revealed that interferon-γ directly triggers proliferative activity and virulence phenotype in pathogenic mycobacteria that allow them to survive and grow inside macrophages. These findings suggest that pathogenic mycobacteria may have evolved eukaryotic-like signal transduction mechanisms to recognize host-protective immune activation.
