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

David Krones,Martin Fraunholz,Carolin Sehl,Katrin Anne Becker,Marcel Rühling,Erich Gulbins,Tobias C Kunz,Kerstin Paprotka

doi:10.3389/fmicb.2021.694489

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.

Highlights

The opportunistic pathogen Staphylococcus aureus (S. aureus) produces a plethora of toxins of which the pore-forming α-toxin is most investigated
In sensitive target cell membranes, α-toxin monomers assemble to a heptameric pre-pore (Bhakdi and Tranum-Jensen, 1991) and subsequent pore formation can lead to calcium influx and efflux of potassium (Fink et al, 1989; Walev et al, 1993; Kwak et al, 2012), or even larger molecules such as ATP (Gierok et al, 2014), eventually leading to host cell death (Baaske et al, 2016)
The activated metalloprotease leads to degradation of cell barrier proteins in epithelial and endothelial cells, allowing for S. aureus deep tissue penetration (Inoshima et al, 2011; Becker et al, 2018). α-toxin sensitivity of target membranes is further dependent on sphingomyelin, since mutants in sphingomyelin synthetase SGMS1 were identified as α-toxin resistant in a genome-wide CRISPR screen (Virreira Winter et al, 2016) and treatment with the bacterial sphingomyelinase staphylococcal β-toxin abrogated α-toxin sensitivity in epithelial cells HBE16o− (Ziesemer et al, 2019)

Summary

INTRODUCTION

The opportunistic pathogen Staphylococcus aureus (S. aureus) produces a plethora of toxins of which the pore-forming α-toxin is most investigated (e.g., reviewed in Bhakdi and Tranum-Jensen, 1991; von Hoven et al, 2019). Ca2+ influx leads to the recruitment of peripheral lysosomes to the plasma membrane, which in turn causes exocytosis of membrane repair enzymes such as lysosomal ASM This process is accompanied by shortterm externalization of lysosomal-associated membrane protein 1 (LAMP1) (Reddy et al, 2001). We here show that treatment with staphylococcal α-toxin induces the recruitment of LAMP1 positive vesicles to the extracellular environment of endothelial cells, which thereby release the membrane repair enzyme ASM into the extracellular environment. This recruitment coincides with elevation of cytosolic calcium levels suggesting that α-toxin triggers membrane repair mechanisms in host cells as they were observed for other pore-forming toxins

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DATA AVAILABILITY STATEMENT