Deciphering the Role of PIG1 and DHN-Melanin in Scedosporium apiospermum Conidia

Abstract

Scedosporium apiospermum is a saprophytic filamentous fungus involved in human infections, of which the virulence factors that contribute to pathogenesis are still poorly characterized. In particular, little is known about the specific role of dihydroxynaphtalene (DHN)-melanin, located on the external layer of the conidia cell wall. We previously identified a transcription factor, PIG1, which may be involved in DHN-melanin biosynthesis. To elucidate the role of PIG1 and DHN-melanin in S. apiospermum, a CRISPR-Cas9-mediated PIG1 deletion was carried out from two parental strains to evaluate its impact on melanin biosynthesis, conidia cell-wall assembly, and resistance to stress, including the ability to survive macrophage engulfment. ΔPIG1 mutants did not produce melanin and showed a disorganized and thinner cell wall, resulting in a lower survival rate when exposed to oxidizing conditions, or high temperature. The absence of melanin increased the exposure of antigenic patterns on the conidia surface. PIG1 regulates the melanization of S. apiospermum conidia, and is involved in the survival to environmental injuries and to the host immune response, that might participate in virulence. Moreover, a transcriptomic analysis was performed to explain the observed aberrant septate conidia morphology and found differentially expressed genes, underlining the pleiotropic function of PIG1.

Keywords: CRISPR-Cas9; DHN-melanin; PIG1; RNA-seq; Scedosporium apiospermum; cell wall.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Gene cluster of melanin biosynthesis in S. apiospermum (36.1 kb), from the published S. apiospermum genome [30]. The scytalone deshydratase encoding gene is located out of the cluster.

Figure 2

Generation of S. apiospermum ΔPIG1 mutants. (a) Restriction map of the S. apiospermum PIG1 locus and strategy for the construction of the disruption cassette. NcoI restriction sites are identified and the purple line indicates the hybridization site of the probe. The size of the expected fragments is indicated by the arrows. (b) Southern-blot analysis of PIG1 transformant strains. Genomic DNA of all strains (parental and mutant derivatives) were digested by NcoI and probed with a hybridization probe corresponding to a 600-bp fragment including the PIG1 3′ flanking region and 3′ end of the HPH gene. The expected signals were 1700 bp for the parental strains and 1123 bp for the M1 and M2 mutants. Two unexpected bands were observed for the T strain (2411 bp and 1700 bp), suggesting integration of the repair cassette upstream of the PIG1 gene. (c) Expression of PIG1 mRNA by the three HPH-resistant transformants relative to their respective parental strain (i.e., T vs. WT, M1 vs. ΔKU70, and M2 vs. ΔKU70). After reverse transcription, mRNA expression was quantified by quantitative PCR. Data were normalized to the expression of the UBC6 and FIS1 housekeeping genes and are representative of four independent experiments. Significant differences are indicated by asterisks: * p ≤ 0.05 (Mann-Whitney test). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 3

Detection of melanin in parental and mutant S. apiospermum strains. (a) Macroscopic aspect of parental and mutant strains after nine days at 37 °C on PDA medium. WT-TRC indicates the WT strain grown on a PDA plate supplemented with 50 µg/mL tricyclazole as a specific inhibitor of melanin biosynthesis. (b) Melanin quantity per million conidia, extrapolated from a standard curve of synthetic melanin, determined by EPR (Graph representative of at least three independent measurements). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 4

Analysis of the fluorescence labelling of conidia surface carbohydrates with FITC-conjugated lectins. (a,b) Fluorescence intensity of (a) ConA binding to mannosyl and glucosyl residues and (b) WGA binding to N-acetylglucosamine (NAG) residues on the surface of the conidia. Fluorescence analysis was performed using ImageJ (Fiji) software from at least 200 conidia. The graph is representative of three independent experiments. Significant differences are indicated: *** p < 0.001 (Kruskall-Wallis test). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 5

Evaluation of cell-wall thickness and integrity. (a) TEM of ΔKU70 and M1 conidia cell walls. The image is representative of the three transformants. (b) Thickness determined from transmission electron microscopy (TEM) acquisition for 20 acquisitions. (c) Growth in contact with the cell-wall chemical stressors Congo Red and SDS. After an incubation of seven days at 30 °C, the colony diameter was measured and expressed according to the control growth on stress-free PDA. All graphs include results obtained from three independent experiments. Significant differences are indicated: * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskall-Wallis test). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 6

Morphology of conidia. (a) Distribution of conidia morphotypes. At least 200 conidia were measured. (b) Distribution of conidia sizes according to cross-sectional area. At least 200 conidia were measured from images of conidia suspensions acquired with a widefield inverted fluorescence microscope (Olympus IX71) at 20× magnification and analyzed using ImageJ (Fiji) software. (c) Transmission electron microscopy (TEM) of ΔKU70 and M1 conidia. Images are representative of the three transformants. Scale bar: 2 µm. T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 7

Biofilm formation. The total biomass was monitored using a crystal violet assay. Results from three independent experiments are shown. Significant differences are indicated as follows: * p < 0.05, ** p < 0.01 (Kruskall-Wallis test). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 8

Susceptibility of the T and the M1 and M2 deleted strains exposed to exogenous stresses. (a) Conidia survival after exposure to UV-C light. After exposure to a dose of 30 mJ/cm2 at 254 nm, conidia were stained with 25 µM propidium iodide (PI) before being analyzed on a LSRFortessaTM cytometer. The graph presents the percentage survival among exposed FITC+ conidia expressed relative to that of unexposed FITC+ conidia (live control), as follows: 100× ((% PI− FITC+ conidia among unexposed control conidia—% PI−FITC+ conidia among exposed conidia)/(% PI−FITC+ conidia among unexposed control conidia)). (b) Conidia survival after exposure to high temperature. After incubation at 40 °C or 50 °C for 15 min, 200 conidia were incubated on PDA plates at 37 °C for 72 h. The number of colonies reflect the number of live conidia. The graph presents the ratio between the number of colonies recovered after exposure of the conidia to heat and the number of colonies recovered after no heat exposure on PDA plates. (c,d) Growth under oxidative conditions. Mycelial growth in PDB medium supplemented with 200 µM cumene hydroperoxide (c) or 40 µM menadione (d) was monitored when incubated for 40 h at 37 °C by nephelometry. Results are expressed in relative nephelometric units resulting from the measurement of the intensity of scattered light through the wall as an indication of the increasing turbidity due to the mycelia. DMSO was used to dissolve the menadione powder. Graphs present the relative growth calculated as the ratio between the nephelometric signal obtained in the presence of an oxidizing compound and in oxidant-free PDB (growth control). All graphs include results obtained from three independent experiments: * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskall-Wallis test). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 9

Functions of differentially expressed genes (DEGs) in M1 relative to ΔKU70. (a) Distribution of functions or metabolic pathways in DEGs with a known function (N = 198). The 80 DEGs with unknown function are not included. (b) Detailed functions of DEGs involved in an oxidoreduction process.

Figure 10

Fold-induction mRNA values for a selected panel of genes in the T, M1, and M2 strains relative to their parental ΔKU70 strain. Expression levels of target genes were normalized against the expression of the S. apiospermum UBC6 and FIS1 housekeeping genes. Results are expressed as 2−ΔΔCq, referring to the fold induction in the T, M1, and M2 strains relative to the mean quantification cycle obtained with the parental strains. Data are from four biological replicates: * p ≤ 0.05 (Mann-Whitney test). T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.

Figure 11

Phagocytosis, killing, and cytokine release from human macrophages infected with T, M1, and M2 conidia. (a) Percentage of macrophages with adherent or ingested conidia after 2 or 6 h of incubation with FITC-stained conidia (MOI 5:1) at 37 °C in 5% CO2. Graphs present the percentage of FITC+ CD11b+ events among CD11b+ cells (macrophages). (b) Conidia killing after ingestion by macrophages. After 6 h of incubation (MOI 1:1), extracellular conidia were removed by three PBS washes and internalized conidia were released by macrophage lysis (cold water). The graph presents the decrease in conidia viability among ingested conidia relative to the viability of conidia not exposed to macrophages (live control). Conidia viability was determined as the percentage of FITC+ PI– events among FITC+ events (total conidia). The reduction in conidia survival was calculated relative to control conidia as: 100 × ((% of live conidia among control conidia − % of live conidia among cell-exposed conidia)/% of live conidia among control conidia). (c) Cytokine release in culture supernatants from macrophages incubated with conidia for 12 h (MOI 10:1). Concentrations were determined using a 13 plex-bead-based LEGENDplex® multianalyte flow assay. A non-infected (NI) condition was included in each experiment. Data are from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskall-Wallis test) T: transformant derived from the WT strain; M1 and M2: transformants derived from the ΔKU70 strain.