Sphingomyelin biosynthesis is essential for phagocytic signaling during Mycobacterium tuberculosis host cell entry

Tuberculosis, the pathological result of infection by Mycobacterium tuberculosis (M.tb), represents a massive and persistent global health burden with vast overrepresentation in regions with poor access to healthcare. This pathogen survives intracellularly within lung macrophages, and has been shown to extensively reprogram the host lipid compartment to the effect of enhanced survival and immune evasion. Sphingolipids are a class of lipid with extensive functions in a wide variety of cellular contexts, including apoptosis, autophagy, stress response, and inflammation. There remains a significant knowledge gap surrounding the lipid-mediated interactions between M.tb and its host. Our investigation aims to explore the mechanisms through which M.tb harnesses host-derived sphingolipids to gain entry to the host and to survive intracellularly. To this end, we use chemical and genetic perturbations of the sphingolipid de novo synthesis pathway and fluorescence microscopy to show that sphingolipids are required for efficient phagocytosis of M.tb. We furthermore show that the dynamics of phosphoinositides at the phagosomal synapse are perturbed upon blockade of sphingolipid synthesis. Finally, we demonstrate that perturbing the homeostasis of a specific sphingolipid (sphingomyelin) through both enrichment and depletion at the cell surface reduces M.tb uptake. This work represents a first step in building comprehensive knowledge of the mechanisms of host-pathogen interactions in M.tb, and demonstrates a cohort of methodologies that may be applied to a variety of pathogenic contexts.