Membrane Sphingolipids Regulate the Fitness and Antifungal Protein Susceptibility of Neurospora crassa

Abstract

The membrane sphingolipid glucosylceramide (GlcCer) plays an important role in fungal fitness and adaptation to most diverse environments. Moreover, reported differences in the structure of GlcCer between fungi, plants and animals render this pathway a promising target for new generation therapeutics. Our knowledge about the GlcCer biosynthesis in fungi is mainly based on investigations of yeasts, whereas this pathway is less well characterized in molds. We therefore performed a detailed lipidomic profiling of GlcCer species present in Neurospora crassa and comprehensively show that the deletion of genes encoding enzymes involved in GlcCer biosynthesis affects growth, conidiation and stress response in this model fungus. Importantly, our study evidences that differences in the pathway intermediates and their functional role exist between N. crassa and other fungal species. We further investigated the role of GlcCer in the susceptibility of N. crassa toward two small cysteine-rich and cationic antimicrobial proteins (AMPs), PAF and PAFB, which originate from the filamentous ascomycete Penicillium chrysogenum. The interaction of these AMPs with the fungal plasma membrane is crucial for their antifungal toxicity. We found that GlcCer determines the susceptibility of N. crassa toward PAF, but not PAFB. A higher electrostatic affinity of PAFB than PAF to anionic membrane surfaces might explain the difference in their antifungal mode of action.

Keywords: Neurospora crassa; Penicillium chrysogenum; antimicrobial proteins; glucosylceramide; lipidomics; sphingolipids.

FIGURE 1

Schematic overview of the GlcCer pathway in N. crassa. Mutants that were used in this study are framed in blue and their defective enzyme function are indicated. Structures were plotted with ChemBioDraw (ChemBioOffice).

FIGURE 2

Lipid quantification in the N. crassa GlcCer knockout mutants and wildtype strains. (A) Heatmap of individual quantifiable ceramide species (means of n = 5, for values see Supplementary Data Sheet S1, Supplementary Table S3). (B) Total abundance of quantified lipid classes in each strain, respectively. PC, phosphatidylcholin; PE, phosphatidylethanolamine; PI, phosphatidylinositol; CL, cardiolipin; Cer, ceramide; MIPC, mannosylinositol phosphorylceramide. (C) Schematic depiction of the ceramide synthesis pathway in N. crassa wt and GlcCer mutants Δlac, Δdes-1, Δdes-2, Δsmt, Δgcs based on the results shown in (A). Fatty acid hydroxylation of ceramides by the putative C. albicans scs7 orthologous gene product (NCU03492) of N. crassa. A respective N. crassa scs deletion mutant was not included in this study. Green box, lipid species; Orange ellipse, genes/enzymes; Red framed light green box, lipids from alternative pathway; Red cross, respective gene knock-out; Question mark: putative enzymatic step.

FIGURE 3

Effect of GlcCer depletion on colony formation of N. crassa. Conidia (10³) were point inoculated on Vogel’s agar and grown for 72 h at 25°C. Magnifications of the colonies are presented for each strain.

FIGURE 4

Effect of GlcCer depletion on the formation of uninuclear and multinuclear conidia. Samples were stained for 5 min with Hoechst 33342 before imaging. The white arrow shows an example for uninuclear conidia, the asterisk indicates an example for multinuclear blastoconidia and the arrowhead for multinuclear arthroconidia. BF, Brightfield. Scale bar 20 μm.

FIGURE 5

Effect of GlcCer depletion on germination and CAT-fusion of N. crassa. (A) Phenotypes of 6 h old germlings: 10⁴ conidia/mL were incubated in 0.2 × Vogel’s medium. Asterisks indicate multiple germ tubes in Δlac, Δdes-1, Δdes-2, and Δgcs mutants. (B) CAT-fusion: (5 × 10⁵ conidia/mL were cultivated for 6 h in 0.2 × Vogel’s medium). Black arrows indicate CAT-fusion. Scale bar 30 μm in (A), scale bar 35 μm in (B).

FIGURE 6

Impact of GlcCer depletion on oxidative stress response. Conidia (10³) were point inoculated on Vogel’s agar (0.75%) containing increasing concentrations of oxidative stress inducers menadione (superoxide radical), H₂O₂ (hydroxyl radicals) and tert-butylhydroperoxide (t-BOOH; organic hydroperoxide). Samples were cultivated for 72 h at 25°C.

FIGURE 7

Impact of GlcCer depletion on the AMP susceptibility. (A) Sensitivity toward PAF: conidia were grown in the presence of increasing PAF concentrations (0–1 μM) for 30 h. To visualize the mycelial growth at 0.06 μM PAF (corresponding to the MIC of the wt strain), magnifications of the insets A–F (framed in red) are presented in the lower panel. (B) Sensitivity toward PAFB: conidia were grown in the presence of increasing PAFB concentrations (0–0.25 μM). Scale bar 150 μm and 75 μm.

FIGURE 8

Uptake of BP-PAF and BP-PAFB in germlings of N. crassa mutants. Samples were taken after 12 h of incubation of conidia with BP-labeled AMPs. Co-staining with propidium iodide was performed 10 min before imaging. Images were taken with the same exposition time (1500 ms). (A) Upper panel: Overview of BP-PAF treated germlings; Red framed lower panel left A–F: Magnification of BP-PAF treated germlings; Framed lower panel right A’–F’: Magnification of BP-PAF treated germlings with increased signal intensity. White arrows indicate BP-PAF uptake into intracellular compartments. (B) Overview of BP-PAFB treated samples. BF, Brightfield; BP, BODIPY-labeled proteins; PI, Propidium iodide. Scale bar 50 and 20 μm in (A), scale bar 50 μm in (B).

FIGURE 9

Impact of PAF and PAFB on the surface charge of N. crassa lipid vesicles. Zeta potential (mV) of N. crassa LUVs obtained from lipid extracts of wt (black bars), Δlac (gray bars) and Δgcs (white bars) in the absence and presence of (A) PAF and (B) PAFB. The AMP concentration was used in relation to 50 μM of LUVs and is expressed as lipid-to-protein molar ratio (L:P). Results are means of 30 measurements in two replicates.