Transcriptome Study of an Exophiala dermatitidis PKS1 Mutant on an ex Vivo Skin Model: Is Melanin Important for Infection?

Abstract

The black yeast Exophiala dermatitidis is a polyextremophilic human pathogen, especially known for growing in man-made extreme environments. Reported diseases caused by this fungus range from benign cutaneous to systemic infections with 40% fatality rate. While the number of cases steadily increases in both immunocompromised and immunocompetent people, detailed knowledge about infection mechanisms, virulence factors and host response are scarce. To understand the impact of the putative virulence factor melanin on the infection, we generated a polyketide synthase (PKS1) mutant using CRISPR/Cas9 resulting in a melanin deficient strain. The mutant and the wild-type fungus were inoculated onto skin explants using an ex vivo skin organ culture model to simulate in vivo cutaneous infection. The difference between the mutant and wild-type transcriptional landscapes, as assessed by whole RNA-sequencing, were small and were observed in pathways related to the copper homeostasis, cell wall genes and proteases. Seven days after inoculation the wild-type fungus completely colonized the stratum corneum, invaded the skin and infected keratinocytes while the mutant had only partially covered the skin and showed no invasiveness. Our results suggest that melanin dramatically improves the invasiveness and virulence of E. dermatitidis during the first days of the skin infection.

Keywords: Exophiala dermatitidis; PKS1; black yeast; melanin; skin model; virulence.

FIGURE 1

Mutated site in the CRISPR/Cas9-targeted gene. The local PKS1 amino acid and nucleotide sequence in the WT is shown at the top (A). The sequence of the reference genome is shown in the middle, while the assembled sequence of the mutant is shown at the bottom of the figure. The orange and mauve rectangles highlight the PAM and deleted sequences, respectively. The mutation leads to a stop codon 7 amino acids downstream of the deleted region (red). Culture of the mutant and wild-type strains (B).

FIGURE 2

HE stained sections of inoculated ex vivo skin explants. Ex vivo skin explants after 2 days (A,C) and 7 days (B,D) inoculated with the wild-type (A,B) and the mutant (C,D) at 37°C. The brown color in (C) and (D) derives from surgical ink used for incisional markings in plastic-reconstructive surgery and does not originate from the mutant fungus. HE stained sections of ex vivo skin explants without fungal inoculum (E,J), the wild-type (F,G,K,L) and the mutant (H,I,M,N). Epidermal samples of the wild-type (F,K) and the mutant (H,M) and dermal tissue of wild-type (G,L) and mutant (I,N) are shown. Areas marked with a black box (E–I) are shown in higher magnification in the lane below (J–N). Scale bars: 100 μm.

FIGURE 3

Upset plot for the sets of enriched annotation for the skin experiments. Set of enriched annotations found in the WT1, WT2, and MT. U and D represent the set of enriched annotation for the up- and down-regulated genes, respectively.