Project description:Transcriptional response of THP-1 cells infected with Mycobacterium tuberculosis utilizing ‘Active’ Mtb and ‘Dormant’ Mtb infection models at different time points. Analysis of the transcriptomic data deciphered the perturbation of gamut of host cellular pathways that are common and differentially manifested in the ‘Active’ Mtb and ‘Dormant’ Mtb infection models.
Project description:To study the role of WhiB3 in regulating host transcriptome, THP-1 were activated with PMA and infected with Mtb and MtbΔwhiB3, host transcriptome was studied.
Project description:The purpose of this study was to identify Mtb- and hsa-encoded miRNAs produced in infected macrophages. RNA from 9 THP-1 samples (3 were uninfected, 3 were infected with Mtb H37Rv for 3 days and 3 were infected with Mtb H37Rv for 6 days) was sequenced and miRNAs were detected.
Project description:This study aims to identify the specific miRNA of mycobacterium tuberculosis (M.tb) infected THP-1 by next-generation sequencing, and further to explore the role of miRNA in innate immunity against M.tb infection.Comprehensive analysis of the next-generation sequencing results showed that the expression of miR-99a-5p was significantly lower in the MTB infected THP-1 cells.
Project description:To compare gene expression changes induced by infection with Mycobacterium tuberculosis (Mtb) with changes induced by purified Mtb products, we infected THP-1 cells with Mtb strain H37Rv or treated with purified Mtb products, then performed RNAseq.
Project description:We examined the microRNA profiles of THP-1 macrophages upon the MTB infection of (1) Beijing/W and non-Beijing/W clinical strains, and (2) susceptible and multidrug-resistant (MDR-) MTB strains. THP-1 cells were induced differentiation into a macrophage phenotype. Then cells were infected with three MDR (INHR, RIFR) Beijing/W, three sensitive (INHS, RIFS) Beijing/W, three MDR(INHR, RIFR) non-Beijing/W, and three sensitive (INHS, RIFS) non-Beijing/W strains. Total RNA were extracted and transfered into cDNA for miRNA profile analysis. Non-infected cells were used as control.
Project description:Tuberculosis (TB) is the deadliest infectious disease worldwide. One obstacle hindering the elimination of TB is our lack of understanding of host-pathogen interactions. Exosomes, naturally loaded with microbial molecules, are circulating markers of TB. Changes in the host protein composition of exosomes from Mycobacterium tuberculosis (Mtb)-infected cells have not been described, can contribute to our understanding of the disease process, and serve as a direct source of biomarkers or as capture targets to enrich for exosomes containing microbial molecules. Here, the protein composition of exosomes from Mtb-infected and uninfected THP-1-derived macrophages was evaluated by tandem-mass-spectrometry and differences in protein abundances were assessed. Our results show that infection with Mtb leads to significant changes in the protein composition of exosomes. Specifically, 41 proteins were significantly more abundant in exosomes from Mtb-infected cells; 63% of these were predicted to be membrane associated. Thus, we used a novel biotinylation strategy to verify protein localization, and confirmed the localization of some of these proteins in the exosomal membrane. Our findings reveal another important scenario where Mtb could be influencing changes in host cells that unveil new features of the host-pathogen interaction and may also be exploited as a source of biomarkers for TB.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
