Exploration de l'ultrastructure et des compartiments intracellulaires de Mycobacterium tuberculosis à l'aide de la cryo-tomographie électronique

Historically, bacteria were thought to lack subcellular organization. It is now widely accepted that prokaryotes, like eukaryotes, compartmentalize biochemical processes via a highly sophisticated intracellular architecture. This includes bacterial microcompartments (BMCs) and intrabacterial lipid-bound compartments, also named intracytoplasmic membranes (ICMs). The exploration of these compartments has been accelerated by novel electron microscopy methods, particularly cryo-electron tomography (cryo-ET) that enable the visualization of intracellular structures at nanometer-scale resolution. Several bacterial phyla have been shown to form ICMs to spatially organize metabolic processes in response to different stimuli. These structures remodel dynamically, highlighting the versatility of bacteria to adapt and sustain energy production in changing environments. So far, research on ICMs has focused on environmental species, however for bacterial pathogens the impact of environmental constraints during the host-pathogen crosstalk have primarily been studied at the proteome or transcriptome level. The intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that affected an estimated 10.8 million people worldwide in 2023 and causes over 1 million deaths each year. Mtb is able to survive a variety of stressful and dynamic microenvironments throughout the course of infection. Macrophages represent the primary target cells of Mtb. As professional phagocytes, they internalize pathogens within phagosomes, a specific microenvironment that rapidly acidifies around the foreign body. They also acquire specific enzymatic activities, generating a superoxide burst, efficiently killing the pathogen. Studies on the transcriptional response of Mtb inside host phagosomes revealed a genome wide remodeling of the pathogen's metabolism to resist and adapt to the macrophage's hostile environment. Indeed, the long history of co-evolution between Mtb and its host has allowed the bacillus to develop an array of factors that simultaneously allow resistance to phagosomal stresses, while blocking phagosome maturation, limiting the full development of these hostile conditions.Previous transcriptomics studies have shown that host-derived signals trigger a hypoxic gene expression response in Mtb and induces a switch from aerobic respiration to anaerobic nitrate respiration, by using nitrate as an alternative terminal electron acceptor. While most mycobacteria possess a functioning nitrate reductase (NR), Mtb has the highest nitrate-reducing capacities, hinting towards a particular importance of nitrate respiration specifically in Mtb. The regulatory circuits of this metabolic adaptation have remained obscure. Also, whether basal nitrate metabolism occurs under normoxic conditions is not fully understood yet. Interestingly, nitrate per se was previously shown not to be a signal leading to increased expression of NR in the presence of oxygen, but low levels of NR activity were constitutively measured, indicating basal nitrate reduction even in presence of oxygen. Furthermore, Mtb possesses a two-component system (TCS), NarSL (Rv0845; Rv0844c) thought to be involved in nitrate sensing and possibly nitrate metabolism regulation, although a deeper understanding of its function is lacking. ICMs have been described in environmental bacteria as main actors during environmental adaptation processes, namely to facilitate metabolic pathways. The widely spread pathogen Mtb is internalized by macrophages, professional phagocytes whose different intracellular niches represent a challenge for the pathogen from the perspective of environmental adaptation. To explore the dynamically changing cellular architecture of Mtb during these events, we combined cryo-ET with cryo-Focused Ion Beam/Scanning Electron Microscopy (cryo-FIB/SEM), proteomics, transcriptomics, and macrophage infections.