Project description:Mycobacterial transcripts were identified in exosomes released from M.tb infected RAW264.7 macrophages that were not present in uninfected exosomes suggesting export of mycobacterial RNA via exosomes Mycobacterial RNA was used as positive control and RNA from exosomes released from uninfected macrophages was used as negative control
Project description:Investigation to study mRNA transcripts present in exosomes from M.tb infected cells and how they compare to those derived from uninfected cells. Transcripts were also studied in donor macrophages as controls The gene expression study identified unique transcripts as well as differentially expressed transcripts present in exosomes released from infected macrophages
Project description:Transcriptional profiling of M.tb H37Rv cells comparing control wild type H37Rv with H37Rv cells electroporated with constitutive expression plasmid pVV16 expressing ESAT-6 binding peptide SL3. The expression of SL3 makes H37Rv less virulent during ex vivo and BalB/c mice infections, sequesters ESAT-6 inside M.tb cells and cause severe defects in mycobacterial morphology. Goal was to determine the effects of SL3 expression on global H37Rv gene expression.
Project description:In this study, RNA sequencing (Transcriptome sequencing) was employed to investigate the global transcriptome changes in the macrophages during the different strains of M.tb infection. THP-1 cells derived macrophages were exposed to the virulent M.tb strain H37Rv (Rv) or the avirulent M.tb strain H37Ra (Ra), and the M.tb BCG vaccine strain was used as a control. The cDNA libraries were prepared from M.tb infected macrophages and then sequenced.
Project description:Transcriptional profiling of M.tb H37Rv cells comparing control wild type H37Rv with H37Rv cells electroporated with constitutive expression plasmid pVV16 expressing ESAT-6 binding peptide SL3. The expression of SL3 makes H37Rv less virulent during ex vivo and BalB/c mice infections, sequesters ESAT-6 inside M.tb cells and cause severe defects in mycobacterial morphology. Goal was to determine the effects of SL3 expression on global H37Rv gene expression. Two color Experiment,Organism: Mycobacterium Tuberculosis, ilife Discoveries designed Custom Mycobacterium tuberculosis on 8x15k GE Microarray. Two-condition experiment, H37Rv vs. H37Rv/SL3. Biological replicates: 2 biological control H37RV replicates labelled with Cy3, 2 SL3 biological expressing replicates labelled with Cy5.
Project description:we studied the role of exosomes isolated from M.tb infected macrophages in modulating the macrophage response to IFN-γ. Nimblegen microarray gene expression studies were used to compare the suppression of IFN-γ inducible genes by exosomes relative to the virulent strain of M.tuberculosis. Overall our study suggest that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanistic link by which M.tb may exert its suppression of host immune response beyond the infected cell, and implies a physiological role for exosomes in immune surveillance of TB. Macrophages were treated with exosomes, infected with M.tb H37Rv or left untreated for 18 hours followed by +/- IFN-γ for an additional 18 hours. Cells were harvested and RNA was isolated and converted to double stranded cDNA and subsequently labeled and hybridized onto Mus musculus 4×72 Nimblegen microarray using Nimblegen Hybridization system 4 according to manufacturer’s instructions (Roche)
Project description:The immune response against tuberculosis relies, at least in part, on CD4+ T cells. Protective vaccines require the induction of antigen-specific CD4+ T cells via mycobacterial peptides presented by MHC class-II in infected macrophages. We have purified MHC class-I and MHC-II peptides and analysed them by mass spectrometry. We have successfully identified 97 mycobacterial peptides presented by MHC-II and 54 presented by MHC-I, from 76 and 41 antigens, respectively. The sequences of selected peptides were confirmed by spectral match validation and immunogenicity evaluated by IFN-gamma ELISpot against peripheral blood mononuclear cells from volunteers vaccinated with BCG, M.tb latently infected subjects or patients with tuberculosis disease. Three antigens were expressed in viral vectors, and evaluated as vaccine candidates alone or in combination in a murine aerosol M.tb challenge model. When delivered in combination, the three candidate vaccines conferred significant protection in the lungs and spleen compared with BCG alone, demonstrating proof-of-concept for this unbiased approach to identifying novel candidate antigens.
Project description:we studied the role of exosomes isolated from M.tb infected macrophages in modulating the macrophage response to IFN-γ. Nimblegen microarray gene expression studies were used to compare the suppression of IFN-γ inducible genes by exosomes relative to the virulent strain of M.tuberculosis. Overall our study suggest that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanistic link by which M.tb may exert its suppression of host immune response beyond the infected cell, and implies a physiological role for exosomes in immune surveillance of TB.
Project description:To explore the regulatory network of noncoding RNAs after M.tb infection and the role of Rv1759c in the infection process, we collected samples of H37Rv- and H37Rv△1759c-infected macrophages and explored the full transcriptome expression profile. We constructed DE-lncRNA/DE-circRNA-DE-miRNA-DE-mRNA regulatory networks during H37Rv and H37Rv△1759c infection. In addition, we first discovered the close relationship between Rv1759c and chemokines during M.tb infection by comparing the transcription profiles of H37Rv and H37Rv△1759c and bioinformatics analysis. Here, our study comprehensively characterizes the ncRNA and mRNA profiles in macrophages infected with H37Rv and H37Rv△1759c, which provides support and new directions for in-depth exploration of ncRNA and PE/PPE family functions during the infection process.