Aylan, Beren; (2024) The dynamic interplay between the Mycobacterium tuberculosis Type VII Secretion System ESX-1 and autophagy in human macrophages. Doctoral thesis (Ph.D), UCL (University College London).

Text Beren Aylan Final Thesis Submission Revised copy.pdf - Other Access restricted to UCL open access staff until 1 May 2025. Download (60MB)

Abstract

The Type 7 Secretion System (T7SS) ESX-1 from Mycobacterium tuberculosis (Mtb) plays a crucial role in evading host responses such as autophagy. However, the transcriptional regulation of ESX-1 during infection of host macrophages remains elusive. In my PhD thesis, I developed a dual promoter reporter system to analyse the activity of the Mtb ESX-1 system using high throughput and single cell live cell imaging. By analysing the expression of these reporters in infected macrophages, I observed a wide range of heterogeneity suggesting that the expression of ESX-1 is regulated at the single bacterial level. ESX-1 function is linked to autophagy but the role of this host cell pathway in controlling Mtb infection is still poorly understood. I determined the contributions of canonical and non-canonical autophagy in the human induced pluripotent stem cellderived macrophages (iPSDM) model, using a set of Mtb mutants deficient for ESX1. I monitored the replication of Mtb mutants that are either unable to trigger canonical autophagy (ΔesxBA) or unable to block non-canonical autophagy(ΔcpsA) in iPSDM lacking either ATG7 or ATG14. I show that deletion of ATG7 resulted in increased replication of Mtb wild type but not of Mtb ΔesxBA or Mtb ΔcpsA. Furthermore, I showed that deletion of ATG14 resulted in increased replication of Mtb wild type and Mtb ΔesxBA. Using the Mtb reporters, I identified a role for ATG14 in regulating fusion of phagosomes with lysosomes, thereby enabling intracellular bacteria restriction. After defining the role of ATG14, I finally combined the ESX-1 reporters with either ATG14-deficient iPSDM or Mtb lacking the lipids phthiocerol dimycocerosates (PDIM), to explore the impact of the environment on the transcriptional regulation of T7SS during infection. My data show that when Mtb is in a restrictive environment some key factors from the ESX-1 system are transcriptionally upregulated, conversely, when the environment is permissive these factors were downregulated.

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