Bernard, Elliott Michael; (2021) The spatiotemporal dynamics of autophagy during Mycobacterium tuberculosis infection of human induced pluripotent stem cell derived macrophages. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The interaction of macrophages with the intracellular pathogen Mycobacterium tuberculosis (Mtb) is critical in determining disease outcomes. Presently, we lack human macrophage models that are genetically tractable, karyotypically normal, and available in large cell numbers. Thus, new human macrophage models are required to facilitate the study of host-pathogen interactions, especially where genetic manipulation is required. In this thesis I set up and characterise a stem cell derived model of human macrophages (iPSDM). iPSDM recapitulate many known interactions between Mtb and human macrophages. An RNA-seq comparison of the transcriptional response to infection between Mtb WT and the attenuated strain Mtb ΔRD1, which lacks the ESX-1 secretion system, revealed commonalities with previous studies along with a novel, RD1 dependent response at 48 h post infection. In macrophages, the autophagy pathway plays diverse roles in infection with intracellular pathogens, including capturing cytosol invading pathogens and targeting them for lysosomal killing. The role of the autophagy pathway during Mtb infection is still unclear. Live cell imaging in iPSDM revealed, following membrane damage by Mtb, the induction of LC3 positive tubulovesicular structures, which fail to capture the bacteria. Correlative 3D focussed ion beam scanning electron microscopy showed the bacteria in the macrophage cytosol following successful dissociation from autophagic structures. Genetic disruption of the autophagy pathway through the knockout of Atg7 failed to alter intracellular Mtb replication or rescue replication of the attenuated mutants Mtb ΔRD1 and Mtb ΔCpsA, which has previously been shown to be more susceptible to restriction by non-canonical autophagy. On the other hand, knockout of Atg14 resulted in enhanced Mtb replication, likely through an autophagy-independent mechanism. This work validates iPSDM to study hitherto unexplored aspects of human macrophage-Mtb interactions. Moreover, these data revealed novel autophagy dynamics in space and time and evasion mechanisms employed by Mtb to subvert this host defence pathway.

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