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

Anna Huber,Nermina Malanovic,Karl Lohner,Willi Salvenmoser,Laura Kovács,Florentine Marx,Gregor Oemer,Markus A Keller,Johannes Zschocke

doi:10.3389/fmicb.2019.00605

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.

Highlights

Fungi are pioneers in colonizing the most diverse habitats with access to organic and inorganic nutrients that enables a saprophytic and parasitic way of life
We further show that the GlcCer biosynthesis pathway mediates Penicillium chrysogenum antifungal protein (PAF) but not Penicillium chrysogenum antifungal protein B (PAFB) susceptibility of the highly sensitive N. crassa, which suggests that the mode of action differs considerably between these two antimicrobial protein (AMP)
Gene candidates involved in the GlcCer biosynthesis were identified in the N. crassa genome2 using the respective C. albicans genes as queries3

Summary

Introduction

Fungi are pioneers in colonizing the most diverse habitats with access to organic and inorganic nutrients that enables a saprophytic and parasitic way of life. Any alteration of the lipid composition of the plasma membrane might impact the distribution, regulation, activity and signaling function of membrane proteins, with adverse effects on the fungal cell fitness. Our knowledge on GlcCer biosynthesis in fungi is based on investigations on yeasts, such as the human pathogenic Cryptococcus neoformans (Rhome et al, 2007; Munshi et al, 2018) and Candida albicans (Oura and Kajiwara, 2010) and on some few reports on filamentous ascomycetes, such as the human pathogen Scedosporium apiospermium (Rollin-Pinheiro et al, 2014), the model organisms Neurospora crassa (Park et al, 2005) and Aspergillus nidulans (Fernandes et al, 2016), the plant pathogen Fusarium graminearum (Ramamoorthy et al, 2007, 2009) and the post-harvest pathogen Penicillium digitatum (Zhu et al, 2014). Saccharomyces cerevisiae and some other yeast species lack the GlcCer synthesis gene and membranes do not contain this sphingolipid (Saito et al, 2006)

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Results

Conclusion