Project description:Gene expression is evaluated over roughly 1.5 cell cycles in a temperature-sensitive dnaA mutant of M. tuberculosis H37Rv to identify genes with periodic expression, or clusters of genes whose expression peaks at different timepoints correlating with distinct cell-cycle events like chromosome replication, septum formation, divisome assembly, and cytokinesis.
Project description:RNA polymerase (RNAP) binding protein RbpA contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Based on structural and biochemical data, four domains have been identified in the RbpA protein: a N-terminal tail (NTT) domain of unknown function, a core domain (CD) that contacts the RNAP β’ subunit in a recently solved crystal structure, a basic linker (BL) that binds DNA, and a s-interaction domain (SID) that binds group I and group II s-factors. However, limited in vivo studies have been performed in mycobacteria and how the individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We dissected the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis. We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP holoenzyme, indicating that the primary function of the CD is not solely association with the RNAP. We show that RbpA BL and SID are required for stabilization of RPo complexes at the ribosomal RNA rrnAP3 promoter in vitro, while the NTT and CD are dispensable. Finally, we determine that the NTT and CD impact gene expression of a distinct set of genes from that affected by the BL and SID activities. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.
Project description:Some intracellular bacteria are known to cause long-term infections for periods of time that last decades without compromising the viability of the host. Although of critical importance, the changes that intracellular bacteria suffer during this long process of residence in a host cell environment remain obscure. Here, we report an experimental approach to study the adaptations of intracellular mycobacteria forced by a long-term intracellular lifestyle. Long-term infection of host macrophages with mycobacteria was maintained for a period of years. Mycobacteria in the long-term infected macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, preference for glucose as a carbon source and neutral lipids accumulation. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states of bacteria living in fluctuating intracellular environments.
Project description:Purified NK cells were co-cultured with M. bovis BCG or M. tuberculosis H37Rv (1:1) in the presence of IL-2 (100U/ml) or IL-12 (10pg/ml) for 24h before trizol extraction. We used microarrays to detail the global gene expression underlying NK cell activation by mycobacteria. NK cell were isolated from the blood of 6 independent donors and activated with different mycobacteria and cytokines in order to study their transcriptional profiles according to mycobacterial virulence.
Project description:Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wildtype N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression. Keywords: Melanoma, checkpoint, microarry