Dataset Information

Major determinants of airway epithelial cell sensitivity to S. aureus alpha-toxin: Disposition of toxin heptamers by extracellular vesicle formation and lysosomal degradation

ABSTRACT: Alpha-toxin is a major virulence factor of Staphylococcus aureus and plays an important role in S. aureus-induced pneumonia. Different sensitivities toward the toxin of various host cell types have been observed. In this study we investigated internalization of pore-containing areas of the plasma membrane in these cell types as well as potential pathways for heptamer degradation (lysosomal, proteasomal) or disposal (formation of exosomes/microvesicles containing toxin pores). 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 seemed to be the lysosomal pathway while proteasomal degradation did not seem to be relevant. Exosomes prepared from culture supernatants of toxin-exposed S9 cells, contained alpha-toxin as well as low amounts of marker proteins of exosomes and microvesicles. These results indicate that the ability of lysosomal degradation of internalized toxin heptamers may be the most important determinant of toxin-resistance of some types of airway epithelial cells. In this study, we compared the proteomes of rHla treated (90 min), 8 h starvation and, 48 h starvation for the S9 cells by mass spectrometry.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Epithelial Cell

SUBMITTER: christian hentschker

LAB HEAD: Jan-Peter Hildebrandt

PROVIDER: PXD023678 | Pride | 2021-04-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20200228_QEp_CH_Zoolo_90_min_Hla_n1.raw	Raw
	20200228_QEp_CH_Zoolo_90_min_Hla_n2.raw	Raw
	20200228_QEp_CH_Zoolo_90_min_Hla_n3.raw	Raw
	20200228_QEp_CH_Zoolo_Ko_48h_n1.raw	Raw
	20200228_QEp_CH_Zoolo_Ko_48h_n2.raw	Raw

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Publications

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

Möller Nils N Ziesemer Sabine S Hentschker Christian C Völker Uwe U Hildebrandt Jan-Peter JP

Toxins 20210224 3

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) o ...[more]

PMID: 33668237

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Project description:The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion paper, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or non-genotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and non-genotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4h and collected 0h, 4h and 20h post-exposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24h. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as non-genotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24h, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the classifier is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. This experiment examined the whole genome transcriptional response of TK6 cells exposed to 0.1 µM aflatoxin B1 (-S9), 10 µg/ml benzo[a]pyrene (-S9), 7.5 mM dexamethasone (-S9) and 24 µg/ml cisplatin (+S9) for 4 hours followed by a 0h, 4h and 20h recovery in fresh media (4h, 8h, and 24h time points, respectively), in addition to negative controls (±S9), vehicle controls (±S9), a direct-acting positive control, cisplatin (-S9) at 24 μg/ml and a positive control requiring metabolic activation, BaP (+S9) at 10 μg/ml. Each concentration and time point had 3 biological replicates, which were pooled for gene expression analysis. There were a total of 14 samples (28 arrays) included in the final analysis using a two-colour dye swap design.

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Dataset Information

Major determinants of airway epithelial cell sensitivity to S. aureus alpha-toxin: Disposition of toxin heptamers by extracellular vesicle formation and lysosomal degradation

Dataset's files

Publications

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

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