Illuminating host defence against mycobacterial infection: interactions with autophagy and LC3-associated phagocytosis

Despite substantial progress in understanding tuberculosis (TB), the eradication of the TB epidemic is still far from reach. This is largely due to the diversity of the causative agent <em>Mycobacterium tuberculosis</em> (<em>Mtb</em>), as well as the variability in host responses and the environmental factors. Genetic and phenotypic heterogeneity endow <em>Mtb</em> with structural resilience and metabolic flexibility, enabling it to survive in diverse microenvironments and establish a successful infection. <em>Mtb</em> has evolved multiple survival, evasion, and subversion strategies to drive transmission, infection, and disease progression. The initial host response is mediated primarily through innate immune effector cells, including granulocytes and macrophages. These cells employ a range of defence mechanisms to restrict <em>Mtb</em> infection, such as <em>Mtb</em>-containing phagosome damage repair, LC3-associated phagocytosis (LAP), autophagy, apoptosis, and granuloma formation. Infection outcome is determined by the dynamic interactions between host and pathogen, which may result in clearance, asymptomatic latent TB, or active TB. Understanding these host-pathogen interactions is essential for developing a comprehensive approach to TB control. <br> The closely related pathogen <em>Mycobacterium marinum</em> (<em>Mm</em>) shares key virulence mechanisms with <em>Mtb</em> and serves as a valuable experimental model to investigate aspects of the host-pathogen interplay that are less accessible using conventional <em>Mtb </em>models. In particular, <em>Mm</em> infection allows real-time analysis of infected host cells' behaviour during early disease stages. In this thesis, we use <em>Mm</em> infection in <em>Danio rerio</em> (zebrafish) larvae as a model to investigate early host-pathogen interactions, with a particular focus on autophagy-mediated degradation. <br> By combining chemical and genetic interventions with advance 3D image analysis in the zebrafish-<em>Mm</em> infection model this work investigates: (i) early host-pathogen interactions between <em>Mm</em> and the autophagic machinery in macrophages; (ii) the role of the lipid kinase PIKfyve in the maturation of <em>Mm</em>-containing vesicles; and (iii) the function of Rubicon, a key regulator linking autophagy and LAP, in host defence against mycobacterial infection. <br> This thesis demonstrates that PIKfyve promotes maturation of <em>Mm</em>-containing vesicles in the autophagy pathway, a process necessary for lysosomal fusion and bacterial degradation. In addition, Rubicon is identified as a critical factor likely facilitating phagosome maturation through LAP activation. Together, these findings advance our understanding of autophagy-related defence mechanisms during early mycobacterial infection in a living host. Importantly, pharmacological stimulation of PIKfyve or Rubicon may provide a basis for host-directed therapies against mycobacterial infections.<br>