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Major Determinants of Airway Epithelial Cell Sensitivity to S. aureus Alpha-Toxin: Disposal of Toxin Heptamers by Extracellular Vesicle Formation and Lysosomal Degradation.

ABSTRACT: Alpha-toxin is a major virulence factor of Staphylococcus aureus. Monomer binding to host cell membranes results in the formation of heptameric transmembrane pores. Among human model airway epithelial cell lines, A549 cells were most sensitive toward the toxin followed by 16HBE14o^- and S9 cells. In this study we investigated the processes of internalization of pore-containing plasma membrane areas as well as potential pathways for heptamer degradation (lysosomal, proteasomal) or disposal (formation of exosomes/micro-vesicles). The abundance of toxin heptamers upon applying an alpha-toxin pulse to the cells declined both in extracts of whole cells and of cellular membranes of S9 cells, but not in those of 16HBE14o^- or A549 cells. Comparisons of heptamer degradation rates under inhibition of lysosomal or proteasomal degradation revealed that an important route of heptamer degradation, at least in S9 cells, seems to be the lysosomal pathway, while proteasomal degradation appears to be irrelevant. Exosomes prepared from culture supernatants of toxin-exposed S9 cells contained alpha-toxin as well as low amounts of exosome and micro-vesicle markers. These results indicate that lysosomal degradation of internalized toxin heptamers may be the most important determinant of toxin-resistance of some types of airway epithelial cells.

SUBMITTER: Moller N

PROVIDER: S-EPMC7996177 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Staphylococcus aureus secretes phenol-soluble modulins (PSMs), a family of small, amphipathic, secreted peptides with multiple biologic activities. Community-acquired S. aureus strains produce high levels of PSMs in planktonic cultures, and PSM alpha peptides have been shown to augment the release of extracellular membrane vesicles (MVs). We observed that amyloids, aggregates of proteins characterized by a fibrillar morphology and stained with specific dyes, co-purified with MVs harvested from cell-free culture supernatants of community-acquired S. aureus strains. δ-toxin was a major component of amyloid fibrils that co-purified with strain LAC MVs, and δ-toxin promoted the production of MVs and amyloid fibrils in a dose-dependent manner. To determine whether MVs and amyloid fibrils were generated under in vivo conditions, we inoculated mice with S. aureus harvested from planktonic cultures. Bacterial MVs were isolated and purified from lavage fluids recovered from infected animals. Although δ-toxin was the most abundant PSM in lavage fluids, amyloid fibrils could not be detected in these samples. Our findings expand our understanding of amyloid fibril formation in S. aureus cultures, reveal the important roles of δ-toxin in amyloid fibril formation and MV biogenesis, and demonstrate that MVs are generated in vivo in a staphylococcal infection model.IMPORTANCEExtracellular membrane vesicles (MVs) produced by Staphylococcus aureus in planktonic cultures encapsulate a diverse cargo of bacterial proteins, nucleic acids, and glycopolymers that are protected from destruction by external factors. δ-toxin, a member of the phenol soluble modulin family, was shown to be critical for MV biogenesis. Amyloid fibrils co-purified with MVs generated by virulent, community-acquired S. aureus strains, and fibril formation was dependent on expression of the S. aureus δ-toxin gene (hld). Mass spectrometry data confirmed that the amyloid fibrils were comprised of δ-toxin. Although S. aureus MVs were produced in vivo in a localized murine infection model, amyloid fibrils were not observed in the in vivo setting. Our findings provide critical insights into staphylococcal factors involved in MV biogenesis and amyloid formation.

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Major Determinants of Airway Epithelial Cell Sensitivity to S. aureus Alpha-Toxin: Disposal of Toxin Heptamers by Extracellular Vesicle Formation and Lysosomal Degradation.

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