Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health burden, with the emergence of antibiotic-resistant strains posing a threat to progress against the disease. Understanding the molecular mechanisms by which Mtb evades the innate immune system and establishes a replicative niche inside macrophages is essential. This thesis utilizes the infection model system consisting of Dictyostelium discoideum (Dd) and Mycobacterium marinum (Mm), providing a powerful and experimentally tractable platform. Dd, a social amoeba, shares characteristics with macrophages as a professional phagocyte, employing a conserved set of bactericidal mechanisms, membrane repair machinery, and degradation pathways to combat intracellular pathogens. Mm, closely related to Mtb, is a fish pathogen and opportunistic human pathogen, employing virulence mechanisms shared to large extent with Mtb. There is a growing consensus that combating antibiotic resistant Mtb strains requires alternative approaches beyond antibiotics. Novel strategies include antivirulence compounds and host-directed therapy (HDT).
Therefore, one aim of this thesis is to enhance our understanding of mycobacterial virulence strategies and host defence mechanisms against intracellular infection. This goal is pursued through two genome-wide approaches: a transposon insertion sequencing (Tn-Seq) experiment to identify genetic requirements for Mm virulence and a large, time-resolved RNA sequencing (RNAseq) experiment to monitor the transcriptomic response of Dd to Mm infection. Notably, Mm genes related to the ESX-1 secretion system, a well-characterised virulence factor, as well as central metabolism and DNA repair, were implicated in intracellular fitness, providing insights for antivirulence strategies. Transcriptomic analysis of Dd revealed stage-dependent responses highlighting pathways crucial for membrane repair, as well as signatures related to the heat-shock response and cell-cycle control as relevant during infection, thus identifying potential targets for host-directed drug intervention.
The development of infection assays involved adaptations to high-throughput formats and benchmarking of antibiotic activities. Additionally, compounds selectively impacting intracellular Mm were benchmarked, expanding the project's scope to include host-defence boosting and antivirulence compounds. Screening of compounds from natural sources, such as a small library of stilbene dimers and a large library of plant extracts, yielded potential antimycobacterial molecules, including stilbene dimers and other polyphenols like honokiol. Recent and preliminary findings suggest that honokiol may affect lipid metabolism and autophagy, warranting further exploration for HDT. However, challenges such as genetic and mechanistic target identification persist.
Future directions involve integrating genome-wide approaches with phenotypic assays for target elucidation. Additionally, advanced techniques such as Limited Proteolysis Mass Spectrometry (LiP-MS) and in silico docking hold promise. Overall, the thesis presents a comprehensive approach to dissecting and manipulating mycobacterial pathogenesis in phagocytes and identifies potential therapeutic candidates.
