Repairing the Breach: Uncovering ER-dependent Membrane Repair during Mycobacterial Infection

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Title: Repairing the Breach: Uncovering ER-dependent Membrane Repair during Mycobacterial Infection
Authors: Anand, Aby
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Thesis advisor: Prof. Dr. Caroline Barisch
Thesis referee: Prof. Dr. Michael Hensel
Abstract: Tuberculosis is one of the top deadliest infectious diseases. Its causative agent, Mycobacterium tuberculosis, employs numerous strategies to survive and persist in macrophages. It induces membrane damage to reduce the endolysosomal toxicity and to exit the Mycobacterium-containing vacuole (MCV). Lesions in the MCV trigger host membrane repair machineries that restore its function and prevent the escape of bacteria. Recently, an ER-dependent repair of damaged lysosomes that is fostered by lipid transfer proteins of the oxysterol binding protein (OSBP) family was described. However, these membrane damage-repair events and especially the role of this pathway during M. tuberculosis infection are largely unexplored. To reveal the underlying molecular mechanisms, I employed the Dictyostelium discoideum/ M. marinum system. In the first part of this thesis, I established 3D-HPF/FS-CLEM together with the electron microscopy facility. This technique is robust, preserves membranes in their utmost native state and provides near-atomic axial resolution. Importantly, it allows envisioning organelle interactions and exit events of mycobacteria in D. discoideum. In the second part of this thesis, I uncovered the formation and function of ER-dependent repair pathway during mycobacterial infection. I found that the ER-Golgi protein OSBP8 re-localizes to the MCV as well as to cytosolic mycobacteria. Strikingly, the mobilisation of OSBP8 is dependent on the presence of the mycobacterial type VII secretion system ESX-1 and presumably on MCV damage. Using 3D-HPF/FS-CLEM and other advanced imaging approaches, I observed that ruptured MCVs recruit ER-tubules containing OSBP8 in its close proximity. Exploring the functional implications of ER-MCV contact sites revealed that depletion of OSBP8 leads to hyperaccumulation of phosphatidylinositol 4-phosphate PI4P on the MCV/lysosomes suggesting OSBP8 balances PI4P on damaged membranes. In addition to this, sterol accumulation in the MCV was slightly reduced. Next, by employing fluorescent probes I observed decreased lysosomal acidification and proteolytic activity in the OSBP8 knockout cells, which promoted mycobacterial growth. Conversely, I observed a growth restriction of mycobacteria in cells overexpressing OSBP8. Next, the conservation of the ER-dependent repair pathway in M. tuberculosis infected macrophages was tested. Remarkably, in agreement to OSBP8 mobilisation, the ER-Golgi protein OSBP re-localized to intracellular mycobacteria in human macrophages in an ESX-1-dependent manner. Taken together, these findings provide the evidence the ER-dependent membrane repair contributes to host resistance against intracellular pathogens including M. tuberculosis.
Subject Keywords: Membrane repair; vacuole escape; Mycobacterium tuberculosis; macrophages; Dictyostelium discoideum; Mycobacterium marinum; oxysterol binding protein; membrane contact site; lysosome; phosphatidylinositol-4-phosphate; sterol
Issue Date: 4-Oct-2023
License name: Attribution 3.0 Germany
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Type of publication: Dissertation oder Habilitation [doctoralThesis]
Appears in Collections:FB05 - E-Dissertationen

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