Sphingomyelin Depletion from Plasma Membranes of Human Airway Epithelial Cells Completely Abrogates the Deleterious Actions of S. aureus Alpha-Toxin.

Sabine Ziesemer,Achim Beule,Jan-Peter Hildebrandt,Christian Müller,Nils Möller,Andreas Nitsch

doi:10.3390/toxins11020126

Abstract

Interaction of Staphylococcus aureus alpha-toxin (hemolysin A, Hla) with eukaryotic cell membranes is mediated by proteinaceous receptors and certain lipid domains in host cell plasma membranes. Hla is secreted as a 33 kDa monomer that forms heptameric transmembrane pores whose action compromises maintenance of cell shape and epithelial tightness. It is not exactly known whether certain membrane lipid domains of host cells facilitate adhesion of Ha monomers, oligomerization, or pore formation. We used sphingomyelinase (hemolysin B, Hlb) expressed by some strains of staphylococci to pre-treat airway epithelial model cells in order to specifically decrease the sphingomyelin (SM) abundance in their plasma membranes. Such a pre-incubation exclusively removed SM from the plasma membrane lipid fraction. It abrogated the formation of heptamers and prevented the formation of functional transmembrane pores. Hla exposure of rHlb pre-treated cells did not result in increases in [Ca2+]i, did not induce any microscopically visible changes in cell shape or formation of paracellular gaps, and did not induce hypo-phosphorylation of the actin depolymerizing factor cofilin as usual. Removal of sphingomyelin from the plasma membranes of human airway epithelial cells completely abrogates the deleterious actions of Staphylococcus aureus alpha-toxin.

Highlights

Airway epithelia form major barriers between inhaled air and the internal space of the body [1].In vivo, respiratory epithelia are covered by a thick mucus layer
Pre-Treatment of Airway Cells with rHlb Allows rHla Monomer Binding to the plasma membranes (PM), but Prevents
To investigate whether SM is necessary for binding of Hla monomers to the host cell plasma membrane or assembly of heptameric transmembrane pores in these membranes, we pre-treated membrane or assembly of heptameric transmembrane pores in these membranes, we pre-treated confluent cell layers (16HBE14o- and S9) for 1 h with 5000 ng/mL rHlb followed confluent cell layers (16HBE14o- and S9) for 1 h with 5000 ng/mL rHlb followed by a 0–4 h incubation with 2000 ng/mL rHla

Summary

Introduction

Airway epithelia form major barriers between inhaled air and the internal space of the body [1].In vivo, respiratory epithelia are covered by a thick mucus layer. Toxins 2019, 11, 126 particles stick to that mucus layer and are removed from the airways by the ciliary activity in the periciliary liquid (mucociliary clearance) [2]. It is unlikely that inhaled bacteria like the human commensal and opportunistic pathogen Staphylococcus aureus (S. aureus) readily come into direct contact with the apical surfaces of epithelial cells. When the mucociliary clearance is attenuated (as in bedridden or immune deprived patients, or patients with virus infections or cystic fibrosis) bacteria may reach critical densities in the mucus layer and start to secrete soluble virulence factors. Virulence factors play a central role in the pathogenicity of S. aureus [3]. Secreted soluble virulence factors like alpha-toxin (hemolysin A, Hla) may diffuse through the mucus layer and reach the apical surfaces of the epithelial cells [4]. The assumption that Hla may play a role in the onset of S. aureus lung infection is supported by the findings of pneumonia patients having generated antibodies against

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Conclusion