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Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles.

Current biology : CB (2020-07-11)
Cara J Ellison, Wanda Kukulski, Keith B Boyle, Sean Munro, Felix Randow
ABSTRACT

Pathogenic 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 [2-4]. 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 [7-9]. 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
OptiPrep Density Gradient Medium, used for cell and subcellular organelle isolation
Sigma-Aldrich
Isopropyl β-D-1-thiogalactopyranoside, ≥99% (TLC)
Sigma-Aldrich
Cholesterol, from sheep wool, ≥92.5% (GC), powder
Sigma-Aldrich
Poly(ethylene glycol), BioUltra, 1,000