Staphylococcus aureus α-Toxin Induces Acid Sphingomyelinase Release From a Human Endothelial Cell Line

Abstract

Staphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

Recovery of fibroblasts from membrane attack byS. aureusα-toxin does not depend on acid sphingomyelinase but involves macropinocytosis

“…Influx of Ca 2+ is considered the principle trigger of PM repair (Cooper & McNeil, 2015). S. aureus α-toxin may increase [Ca 2+ ] i. in certain cell types, for instance lung epithelial cells (Eichstaedt et al, 2009), keratinocytes (HaCaT cells), (von Hoven et al, 2016), and endothelial cells (Krones et al, 2021). Yet, it is not clear whether these increases are due to Ca 2+ influx through toxin pores, other channels, or some secondary lesions caused by endogenous PFP, or through ruptures.…”

“…On the other hand, there is evidence, that membrane repair after damage by S. aureus α-toxin depends on different mechanisms (Husmann et al, 2009) (Husmann et al, 2006), , (Valeva et al, 2000). However, according to a recent report, α-toxin may trigger release of ASM from cells (Krones et al, 2021), although the functional significance of this finding is not known. In order to clarify whether ASM is a requirement for membrane repair after attack by α-toxin we decided to investigate fibroblasts lacking functional ASM.…”

“…At least in some cell types, comparably high concentrations of α-toxin may cause moderate increases of cellular [Ca 2+ ] i (Walev et al, 1993) (von Hoven et al, 2016), but it is unclear whether this is due to ion flux through α-toxin pores of some ill defined high conductance conformation, or through cellular channel forming proteins, possibly including endogenous PFP like gasdermins, or through rupturesof the PM (von Hoven et al, 2019). A potential role of calcium-influx and ASM for membrane repair in endothelial cells treated with α-toxin was implicated by a recent report, that α-toxin induces release of this enzyme (Krones et al, 2021), but to our knowledge, there is no direct experimental evidence for ASM-dependent repair in response to α-toxin. Here we addressed the question whether ASM is required for membrane repair after attack by S. aureus α-toxin.…”

Functional roles of sphingolipids in immunity and their implication in disease

“…Most pathogenic bacteria and viruses act as “parasites” of host sphingolipid metabolism, thereby disturbing the host defense system 16 , 53 , 65 . Neisseria gonorrhoeae , the major cause of meningitis and septicemia, Pseudomonas aeruginosa , which is an opportunistic pathogen and the cause of cystic fibrosis and sepsis, and Staphylococcus aureus can stimulate the acid SMase (aSMase) of epithelial cells and fibroblasts, facilitating invasion through a sphingomyelin-decreased and ceramide-enriched platform 97 – 99 and evading the innate immune defense system 100 – 102 . The enrichment of ceramides in host cells can induce cell death and thereby destroy the barriers formed by cells.…”

“…The enrichment of ceramides in host cells can induce cell death and thereby destroy the barriers formed by cells. Even worse, several kinds of toxins produced by pathogens (e.g., the α-toxin produced by S. aureus ) can bind to surface metalloproteinases (e.g., ADAM10) of the host and induce the release of acid ceramidase-mediated ceramide outside of the inflamed walls, which eventually leads to degradation of tight junctions 99 . Moreover, the produced chemokines and S1P recruit innate/adaptive immune cells, and recruited macrophages can wisely utilize the SMase/ceramidase strategy of the pathogen to rearrange its cytoskeleton, facilitating the process of phagocytosis 96 , 103 .…”