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The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.