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. 2018 Jun 15;8(1):9182.
doi: 10.1038/s41598-018-27451-9.

Extracellular Nanovesicles Released From the Commensal Yeast Malassezia Sympodialis Are Enriched in Allergens and Interact With Cells in Human Skin

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Free PMC article

Extracellular Nanovesicles Released From the Commensal Yeast Malassezia Sympodialis Are Enriched in Allergens and Interact With Cells in Human Skin

Henrik J Johansson et al. Sci Rep. .
Free PMC article

Erratum in

Abstract

Malassezia sympodialis is a dominant commensal fungi in the human skin mycobiome but is also associated with common skin disorders including atopic eczema (AE). M. sympodialis releases extracellular vesicles, designated MalaEx, which are carriers of small RNAs and allergens, and they can induce inflammatory cytokine responses. Here we explored how MalaEx are involved in host-microbe interactions by comparing protein content of MalaEx with that of the parental yeast cells, and by investigating interactions of MalaEx with cells in the skin. Cryo-electron tomography revealed a heterogeneous population of MalaEx. iTRAQ based quantitative proteomics identified in total 2439 proteins in all replicates of which 110 were enriched in MalaEx compared to the yeast cells. Among the MalaEx enriched proteins were two of the M. sympodialis allergens, Mala s 1 and s 7. Functional experiments indicated an active binding and internalization of MalaEx into human keratinocytes and monocytes, and MalaEx were found in close proximity of the nuclei using super-resolution fluorescence 3D-SIM imaging. Our results provides new insights into host-microbe interactions, supporting that MalaEx may have a role in the sensitization and maintenance of inflammation in AE by containing enriched amounts of allergens and with their ability to interact with skin cells.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Cryo-electron tomography of MalaEx. (A) Central section of a tomographic reconstruction of MalaEx harvested from M. sympodialis cultured for 48 h in RPMI medium. The full tomogram is shown in Movie 1 (see Supplementary information). (B) Zoom into the green box of A showing a low electron density vesicle (red arrow) and high electron density vesicles with coated surfaces (yellow arrows). (C) Isosurface representation of the vesicles enclosed by the blue box in A. The surface representation shows two small vesicles with low electron density (in red) encircled by a larger one (in grey).
Figure 2
Figure 2
Characterizing MalaEx proteins by proteomics. (A) Quantitative overview of iTRAQ based proteomics experiments to define MalaEx enriched proteins by comparing MalaEx to M. sympodialis whole cells (WC). R 1–4 denotes the 4 biological replicates cultured for 72 h in mDixon broth, and 2439 proteins with quantification across all 8 samples are shown. (B) Volcano plot to define proteins enriched in MalaEx compared to WC. Proteomics data as in (A). Vertical and horizontal dashed lines indicate 95 percentile of ratios and q-value < 0.1, respectively. Mala s allergens are indicated in red. (C) Venn diagram depicting overlap between proteins considered enriched in MalaEx compared to WC. (D) Gene ontology (GO) analysis of MalaEx. GO term distribution in proteins defined as enriched in MalaEx (from AC) compared to all identified proteins. Numbers indicate the number of M. sympodialis proteins that could be mapped to yeast homologs for the analysis. Arrows highlight proteins most enriched in MalaEx.
Figure 3
Figure 3
Cellular uptake of MalaEx in human primary keratinocytes and monocytes. (AH) Confocal microscopy images following incubation of MalaEx, harvested from M. sympodialis cultured for 48 h in RPMI medium and Vybrant Dil labeled (red; 10 µg/ml), with (A,C) keratinocytes stained for E-cadherin (green) and (B,D) monocytes stained for CD14 (green) at 37 °C and 4 °C for 2 h and 16 h. Arrows highlight cells interacting with MalaEx. (E) Keratinocytes treated with Vybrant Dil control for 16 h at 37 °C and (F) cultured alone. (G) Monocytes treated with Vybrant Dil control for 16 h at 37 °C and (H) cultured alone. (I; left image) Structured Illumination Microscopy (3D-SIM) of keratinocytes incubated with MalaEx for 16 h at 37 °C from a single axial slice (depth 116 nm). (I; right image). Zoomed in 3D-SIM image of selected area (white square in left image) showing perinuclear localized MalaEx. Dashed line indicates the borderline between the nuclei and the cytoplasm of the keratinocyte.

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