Down‐regulation of acid sphingomyelinase and neutral sphingomyelinase‐2 inversely determines the cellular resistance to plasmalemmal injury by pore‐forming toxins

Abstract

Bacterial pore‐forming toxins compromise plasmalemmal integrity, leading to Ca2+ influx, leakage of the cytoplasm, and cell death. Such lesions can be repaired by microvesicular shedding or by the endocytic uptake of the injured membrane sites. Cells have at their disposal an entire toolbox of repair proteins for the identification and elimination of membrane lesions. Sphingomyelinases catalyze the breakdown of sphingomyelin into ceramide and phosphocholine. Sphingomyelin is predominantly localized in the outer leaflet, where it is hydrolyzed by acid sphingomyelinase (ASM) after lysosomal fusion with the plasma membrane. The magnesium‐dependent neutral sphingomyelinase (NSM)‐2 is found at the inner leaflet of the plasmalemma. Because either sphingomyelinase has been ascribed a role in the cellular stress response, we investigated their role in plasma membrane repair and cellular survival after treatment with the pore‐forming toxins listeriolysin O (LLO) or pneumolysin (PLY). Jurkat T cells, in which ASM or NSM‐2 was down‐regulated [ASM knockdown (KD) or NSM‐2 KD cells], showed inverse reactions to toxin‐induced membrane damage: ASM KD cells displayed reduced toxin resistance, decreased viability, and defects in membrane repair. In contrast, the down‐regulation of NSM‐2 led to an increase in viability and enhanced plasmalemmal repair. Yet, in addition to the increased plasmalemmal repair, the enhanced toxin resistance of NSM‐2 KD cells also appeared to be dependent on the activation of p38/MAPK, which was constitutively activated, whereas in ASM KD cells, the p38/MAPK activation was constitutively blunted.—Schoenauer, R., Larpin, Y., Babiychuk, E. B., Drücker, P., Babiychuk, V. S., Avota, E., Schneider‐Schaulies, S., Schumacher, F., Kleuser, B., Köffel, R., Draeger, A. Down‐regulation of acid sphingomyelinase and neutral sphingomyelinase‐2 inversely determines the cellular resistance to plasmalemmal injury by pore‐forming toxins. FASEB J. 33, 275–285 (2019). http://www.fasebj.org

Keep Your Friends Close, but Your Enemies Closer: Role of Acid Sphingomyelinase During Infection and Host Response

“…Subsequent formation of ceramide-enriched, topically restricted domains in the outer leaflet of the lipid bilayer is an essential step contributing to tighter packing of the membrane, forwarding a negative curvature and inward vesiculation of the damaged area. At the end, the resulting internal degradation contributes to cellular integrity and survival (72,73), e.g., after exposition of cells toward listeriolysin O or pneumolysin (74).…”

Mechanisms protecting host cells against bacterial pore-forming toxins

“…In particular, ASM was shown to hydrolyze sphingomyelin into phosphorylcholine and ceramide domains, triggering the endocytosis of PFT pores within small (50–100 nm) lipid-rich PM invaginations termed caveolae [ 115 – 117 ]. Caveolin- or ASM-deficient cells have impaired ability to repair PFT-induced injuries [ 104 , 118 , 119 ] and extracellular ASM is sufficient to promote SLO internalization [ 104 , 115 ]. However, to date, internalization of active pores by caveolar-dependent mechanisms has never been directly visualized and the role of endocytosis as a mechanism of PFT removal remains controversial.…”

Sphingomyelin Breakdown in T Cells: Role of Membrane Compartmentalization in T Cell Signaling and Interference by a Pathogen

“…On stimulation, the role of NSM2 in T-cell morphology driven by actin reorganization may differ depending on receptor signaling: while NSM2 ablation in primary T cells enhanced spreading responses in response to CD3/CD28 engagement, morphological polarization of CD4 + T cells on fibronectin-coated surfaces and brain endothelial cells was effectively limited (Mueller et al, 2014; Collenburg et al, 2017) (Figure 3). While ASM deficiency did not detectably affect T-cell morphology, its hyperactivation in response to MV caused collapse of actin-based protrusions in ASM-sufficient cells (Gassert et al, 2009; Schoenauer et al, 2019). This could be rescued by ablation of the ASM, but also of the NSM2.…”

“…Morphological polarization requires cytoskeletal dynamics. In the steady state, NSM2 may negatively control actin metabolism in T cells, because the average cell volume and frequencies of hairy-appearing, abundant protrusions are significantly elevated in NSM2-deficient Jurkat cells (Schoenauer et al, 2019). On stimulation, the role of NSM2 in T-cell morphology driven by actin reorganization may differ depending on receptor signaling: while NSM2 ablation in primary T cells enhanced spreading responses in response to CD3/CD28 engagement, morphological polarization of CD4 + T cells on fibronectin-coated surfaces and brain endothelial cells was effectively limited (Mueller et al, 2014; Collenburg et al, 2017) (Figure 3).…”

“…Moreover, production of TCR-containing vesicles and their release into the synaptic cleft have been found to enhance T-cell activation (Choudhuri et al, 2014). Though both NSM2 and ASM are known to regulate both cytoskeletal activity and endo/exocytosis (Draeger and Babiychuk, 2013; Schoenauer et al, 2019) (see also below), their involvement in these particular features of the mature IS has not been revealed as yet. In contrast, NSM-dependent formation and polarized release of exosomes from T cells toward the APC were described, revealing that this enzyme indeed contributes to communication at the level of the IS (Mittelbrunn et al, 2011).…”