Impact of Mycobacterium tuberculosis exosomes

Tuberculosis (TB) is one of the top 10 causes of death worldwide, and 9 million TB cases are diagnosed annually on a worldwide basis. The current epidemic is aggravated by the emergence of more adapted and drug-resistant variants of Mycobacterium tuberculosis (Mtb), the causative agent of the disease. Extracellular vesicles (EVs) are small cell-derived membrane blebs that are released from mosteukaryotic (exosomes) and prokaryotic (membrane vesicles, MVs) cells into their extracellular environment. EVs have recently attracted increasing interest because of their potential function in cell-to-cell communication, in gene therapy delivery and as virulence factors, with implications in several diseases including TB. The main goal of the ExoTB project is to characterize prokaryotic membrane vesicles (MVs) and eukaryotic exosomes in tuberculosis (TB), with the hypothesis that they contribute to intercellular communication among Mtb cells and host cells in TB disease, including meningitis. Therefore we isolated and characterized MVs from Mtb cells and exosomes from TB patients. In order to address how and why MVs escape the thick cell walls of mycobacteria, we characterized the proteomic profile of Mtb to unveil the role of MVs in mycobacterial adaptation to environmental factors. Analysis of the exosomes, whole Mtb cells and purified MVs by mass spectrometry (MS) identified the functional categories of the proteins involved in EVs. The MS data revealed that Mtb MVs contain enriched packaging of virulence associated mycobacterial proteins such as antigen-85 complexes, ESAT-6 and PE/PPE, etc. Other MV proteins identified were found to be involved in two-component systems, ABC transporters, amino acid and fatty acid metabolism. Notably, we identified key signatures of post-translational modifications in MVs versus whole cells. Thereby, we characterized fast and slowly growing Mtb naturally occurring strain variants and their MVs. Then, we analyzed the changes that occurred at the transcriptomic level and discovered a multitude of novel ncRNAs associated with EVs. The ncRNAs play important roles in the regulation of mRNAs and the proteins encoded, providing quick responses to changing environmental conditions. The exosomal ncRNAs responding to infection among TB patients identified provided clues to their critical role in post-transcriptional gene regulation, which may influence the pathogenesis of TB. Microbe-host interactions in TB were investigated in glial cells, macrophages and lymphocytes. We examined the interaction between astrocytes, the most abundant glial cells in the brain, and Mtb, through their exosome and MV structures, focusing on the role of astrocytes in their responses to TB meningitis. The presence and nature of exosomes in glial cells and MVs in Mtb was determined, yielding the lipoprotein LpqH as a relevant biomarker for Mtb MVs. When investigating microbe-host interactions in TB, we characterized the MVs and exosomes detected with a focus on TB meningitis. ExoTB tested the hypothesis that MVs from Mtb trigger cellular responses that predisposes an individual to infection, in glial cells, macrophages and lymphocytes. ExoTB has convergently employed functional ?omics and biochemical approaches in glial cell lines to delinate the meningeal response to TB meningitis. This characterization has mainly focused on the glial response to TB meningitis and Mtb MVs / membrane blebs, with particular emphasis on glial astrocytes. Also, some TB patients exhibit higher amounts of EVs in their plasma than others. The presence of exosomes in glial cells and patient plasma from pulmonary and extra-pulmonary TB patients including TB meningitis were useful in this comparison. The studies of EVs from Mtb and host cells by MS contribute to defining differential protein profiles with emphasis on MV characteristics and antigenicity versus Mtb whole cell lysates. In general, MVs and exosomes have a potential relevance for biomarker discovery, vaccine design and drug delivery. Capacity building: The ExoTB project is a multidisciplinary endeavour that engages scientists from complementary fields of research in a unified action to resolve the role of the membrane blebs. A variety of next-generation molecular and comparative studies are performed to define the physiological role of blebs and their potential use in diagnostics and therapeutics. Our group has provided TB diagnostic, molecular and bioinformatics training for representatives from Ethiopia, Sudan and South Sudan in Ethiopia and in Norway. The clinical and molecular studies and advanced training and capacity building have resulted in technology transfer between industrial and LMIC countries, highly relevant for the GLOBVAC call.

Tuberculosis (TB) is a major public health concern, and 9 million TB cases are diagnosed annually on a worldwide basis. Extracellular vesicles (EV) are small cell-derived membrane vesicles approximately 100 nm in size released from most eukaryotic (exosom es) and prokaryotic (membrane vesicles, MVs) cells into their extracellular environment. EVs have recently attracted interest because of their potential function in cell-to-cell communication, virulence and small RNA delivery, with implications in several diseases, in addition to their extensive use as bacterial vaccines and biomarker potential. The ExoTB project is a multidisciplinary endeavour that will employ functional and comparative genomics, lipidomics, transcriptomics and biochemical approaches, a s well as cellular and mouse model systems, to analyse the global meningeal glial responses in health and TB disease including meningitis. The main goal of this project is to characterize bacterial and eukaryotic EVs, MVs and exosomes, respectively, w ith the hypothesis that they contribute to intercellular communication among Mycobacterium tuberculosis (Mtb) cells and glial astrocytes in the brain. Therefore, we will examine the interaction between astrocytes, the most abundant glial cells in the brai n, and Mtb, through the exosome and MV structures which they form. Next-generation imaging and molecular techniques will be developed and applied, focusing on the role of astrocytes in their responses to TB meningitis. The nature and role of exosomes in g lial cells and MVs in Mtb will be determined. Finally, the potential relevance of MVs and exosomes for biomarker discovery, vaccine design and drug delivery will be explored, highly relevant for the GLOBVAC call.