Deconstructing the cell wall of the tubercle bacillus, brick by brick

Mycobacterium tuberculosis (Mtb) is protected by an unusual cell wall that plays a crucial role in the interaction with the host. However, thus far our knowledge of proteins that functionalize this cell wall is limited. In this project, we will focus on an important but thus far understudied group of proteins of this cell wall, the lipoproteins. The Mtb genome is coding for 99 different putative lipoproteins. Some of them are required for nutrient acquisition or for lipid transport, but the majority has unknown functions. Interestingly, most of these lipoproteins are highly conserved in other mycobacterial pathogens, indicating their importance. Recent advances in mycobacterial genetics and cell imaging techniques allow us to systematically study the role of Mtb lipoproteins. First, we will use the CRISPR/Cas9 technology to introduce frameshift mutations in all genes encoding lipoproteins to generate a collection of lipoprotein-knockout Mtb strains. Then, for each of these strains, we will study (1) Mtb morphology, growth and protein secretion in vitro and (2) the infection of human macrophages, the most important host cell in tuberculosis. We will analyze critical aspects of the host-pathogen interaction, including phagocytosis, activation of the key macrophage signaling pathways, Mtb killing by macrophages and Mtb-induced macrophage death. We will identify those lipoproteins whose knockouts lead to significantly altered responses and will subject them to a more detailed analysis, characterizing their location, their interacting partners in Mtb and the effects on cell envelope composition. Together, this study will uncover key lipoproteins required for the biogenesis of the protective cell envelope of Mtb and will reveal key elements of Mtb for the interaction with the human macrophage. These crucial lipoproteins will be interesting candidates for antibiotic targets and vaccine development.