Characterisation of in vivo Mycobacterium tuberculosis for novel antimicrobial drug discovery

Abstract

Tuberculosis (TB) remains one of the top ten causes of death worldwide and is the leading cause of death by a single bacterial agent with 1.5 million deaths in 2020. The development of new multi-drug therapies is limited by the small number of validated drug targets, therefore novel strategies are required to understand the pathways used by Mycobacterium tuberculosis (M.tb) to survive in lung granulomas where drug treatment is targeted. At present, in vitro models fail to accurately reproduce the localised microenvironments of the tuberculosis lung and most animal models share limited pathology with human disease. Unsurprisingly, subsequent drug candidate and vaccine development has been hampered. Therefore, new models, which more accurately reflect the human tuberculosis lung, are required to screen novel drug candidates. The aims of this project were to use novel approaches to isolate M.tb RNA from lung tissue and characterise how the in vivo M.tb transcriptome differs from axenic culture to facilitate the discovery of novel drug targets. Then, to create an in vitro macrophage model that is more representative of human necrotic lung lesions. M.tb murine lung signatures were defined using differential lysis and RNA-sequencing of M.tb isolated from murine lung tissue. 150 genes were significantly induced after intravenous compared to aerosol route of murine infection, which may reflect variations in lung disease (in this case driven by route of infection) that may impact drug efficacy in the lung. These results have implications for the interpretation of murine studies and drug action in TB patients with diverse lung pathologies. These methodologies were applied to human lung resection samples, where metabolically active M.tb was detected from most TB patients with recurrent symptoms even after treatment completion. Ribosome-sequencing methodologies were developed to explore translation in M.tb, revealing that genes important for bacterial survival were more heavily translated in vitro. The M.tb transcriptional data collected from murine and human lung tissue was interpreted to determine parameters for a new necrotic macrophage infection model using bioluminescence to assay drug efficacy. This project demonstrates that transcriptome analysis of M.tb from lung tissue identifies key pathways expressed during TB disease. The use of more biologically relevant in vitro drug screens could importantly reduce the number of animals used in scientific research and improve the translation of findings from the lab to the clinic.

Date of Award	May 2022
Original language	English
Awarding Institution	University of Brighton
Supervisor	Simon Waddell (Supervisor)