Abstract
SummaryPathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2, 3, 4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6]. The latter may cause membrane damage directly, when inserting their Type Three Secretion needles into host membranes, or indirectly through translocated bacterial effector proteins [7, 8, 9]. Here, we report that sphingomyelin, an abundant lipid of the luminal leaflet of BCV membranes, and normally absent from the cytosol, becomes exposed to the cytosol as an early predictive marker of BCV rupture by Gram-negative bacteria. To monitor subcellular sphingomyelin distribution, we generated a live sphingomyelin reporter from Lysenin, a sphingomyelin-specific toxin from the earthworm Eisenia fetida [10, 11]. Using super resolution live imaging and correlative light and electron microscopy (CLEM), we discovered that BCV rupture proceeds through two distinct successive stages: first, sphingomyelin is gradually translocated into the cytosolic leaflet of the BCV, invariably followed by cytosolic exposure of glycans, which recruit galectin-8, indicating bacterial entry into the cytosol. Exposure of sphingomyelin on BCVs may therefore act as an early danger signal alerting the cell to imminent bacterial invasion.
Highlights
Lysenin Detects Exposure of Sphingomyelin to the Cytosol To visualize whether sphingomyelin becomes exposed to the cytosol upon endomembrane damage, we developed a fluorescent sphingomyelin reporter based on Lysenin, a sphingomyelinbinding member of the aerolysin family of b-pore-forming toxins from the earthworm Eisenia fetida [10, 11]
Sphingomyelin Exposure on Salmonella-containing vacuole (SCV) Is T3SS Dependent We investigated the cause of sphingomyelin exposure on SCVs
Sphingomyelin Is Exposed on Damaged SCVs before Glycans To investigate the process by which Gram-negative bacteria rupture their vacuole en route to the cytosol, we studied Lysenin recruitment to SCVs in the context of galectin-8 as a marker of membrane rupture
Summary
Lysenin Detects Exposure of Sphingomyelin to the Cytosol To visualize whether sphingomyelin becomes exposed to the cytosol upon endomembrane damage, we developed a fluorescent sphingomyelin reporter based on Lysenin, a sphingomyelinbinding member of the aerolysin family of b-pore-forming toxins from the earthworm Eisenia fetida [10, 11]. To enable expression of the otherwise toxic protein, we deployed either the isolated C-terminal doma
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