Targeted correlative light and electron microscopy in the study of host-pathogen interactions

Abstract

Abstract Tuberculosis (TB) and HIV are the two deadliest infectious diseases in the world today. 1,8 million people died from TB in 2015, 400.000 of which were HIV positive. Drug resistant TB strains are rapidly spreading, antibiotic treatment regimens are extensive and current vaccines are inefficient. In this thesis, we aimed to establish new technological approaches for the investigation of intracellular infections, with the ultimate goal of advancing research towards the development of new and more effective treatment and vaccine strategies. TB is caused by the slow-growing tubercle bacillus Mycobacterium tuberculosis (Mtb). It was widely believed for decades that Mtb could uniquely be located within membrane bound phagosomal compartments of its host cell. In recent years however, several studies have demonstrated how Mtb is in contact with and even enters the cytosol of its host cell. Translocation to the cytosol exposes Mtb to detection by cytosolic immune sensors such as inflammasome complexes. Activation of inflammasomes leads to a potent proinflammatory response from the cell, usually accompanied by a lytic form of cell death termed pyroptosis. The mechanisms involved in inflammasome activation and cell death by Mtb remain elusive. In this thesis, we developed advanced microscopy methods to study the intracellular life of mycobacteria in infected macrophages, and employed similar methods to investigate HIV in T cells. We developed a system that enabled streamlined observation of infected single cells by live-cell imaging, confocal microscopy and Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography. FIB/SEM tomography is a method that allows imaging of whole cells in 3D at nanometer resolution. All imaging modalities were applied to the very same cells, called correlative light and electron microscopy (CLEM). This allowed linking ultrastructural information observed at the EM level to protein-specific information from confocal imaging, and to previous events as imaged by live-cell microscopy. In particular, we focused on the details of cytosolic contact between Mtb and their host cell over time, and how this influenced on host cell immune responses. The dynamics of inflammasome activation and pyroptosis induced by Mtb could be delineated, and we imaged, for the first time, the intact Mtb-induced inflammasome in infected macrophages.