Sphingomyelin as a danger signal in cell-autonomous immunity

Individual cells employ mechanisms of cell-autonomous immunity to defend their cytosol against bacterial invasion. One such mechanism involves indirect detection of the pathogen through recognition of pathogen-induced disturbances causing the appearance of specific host molecules in an abnormal location. For example, glycans, which are located on the extracellular leaflet of the plasma membrane under homeostatic conditions, become hidden inside bacteria-containing vacuoles (BCVs) during bacterial entry into the cell. Upon BCV rupture, glycans become exposed to the cytosol where they act as a danger signal and are detected by the cytosolic danger receptor, Galectin 8. My research reveals that sphingomyelin, a host lipid predominantly located on the outer leaflet of the plasma membrane, is exposed to the cytosol on damaged BCVs. I visualised the appearance of intracellular sphingomyelin by utilising Lysenin - a sphingomyelin-specific toxin from earthworms – as a cytosolic sphingomyelin reporter. Lysenin is recruited to BCVs in a sphingomyelin-dependent manner upon cytosolic entry of both Gram-negative and Gram-positive bacteria. Lysenin co-localises with Galectin 8 on a proportion of BCVs, indicating that sphingomyelin exposure occurs upon membrane damage. Moreover, I elucidated that sphingomyelin exposure occurs before glycan exposure on damaged BCVs indicating that BCV rupture may proceed through two stages: ‘minor’ and ‘major’ damage. My investigations into possible causes of vacuole rupture are on going. To identify endogenous cellular receptors for cytosol-exposed sphingomyelin, I established and executed an assay to compare enrichment of mammalian cell lysate proteins on liposomes containing or lacking sphingomyelin. Following mass spectrometry analysis, 49 candidate proteins were tested for recruitment to Salmonella. Twelve candidates were recruited to BCVs upon infection. Of these twelve, I pursued five candidates in greater detail due to their recruitment to Salmonella being either entirely unknown, or known, but via a non-sphingomyelin mechanism. Further analysis of one candidate in particular, TECPR1, elucidated that TECPR1 is recruited to Salmonella in a sphingomyelin-dependent manner and possesses sphingomyelin-specific binding properties in vitro. Therefore, my thesis research identifies TECPR1 as an endogenous sphingomyelin-binding protein