Introduction

The mould Aspergillus fumigatus is an opportunistic human pathogen, which can cause multiple diseases ranging from life-threatening invasive pulmonary aspergillosis in immunocompromised patients to chronic, noninvasive forms of infection and allergies. The ability of the fungus to sense and adapt to low environmental oxygen concentrations is an important virulence trait. In order to study the adaptation response on the protein and posttranslational level, we performed quantitative proteomics and phosphoproteomics analyses by comparing fungal mycelium grown under either normoxic or hypoxic (0.2% O2) conditions in an oxygen-controlled fermenter. The goal was to find novel regulatory circuits in hypoxic adaptation.

Methods

Proteins were extracted from A. fumigatus mycelium and digested in solution with a trypsin/LysC mixture. Subsequently, phosphopeptides were enriched using a TiO₂/ZrO₂ solid phase extraction protocol. All samples were analyzed in triplicates by nanoLC-MS/MS (Thermo QExactive HF). The pTmRS algorithm was used to calculate phosphosite probabilities and the Minora algorithm for label-free quantification.

Results

We identified in total 5136 proteins, of which 318 proteins and 1674 phosphopeptides showed significantly different abundance upon hypoxia (fold change >4, ratio-adjusted p-value <0.05). In particular proteins involved in mitochondrion organization, amino acid metabolism, and lipid metabolic processes increased in abundance under hypoxia. The phosphoproteomic data indicated that the mitotic cell cycle and autophagy processes are differentially regulated under hypoxic growth. Indeed, phosphopeptides derived from proteins of the Atg1/Atg13 complex, which is known to initiate autophagosome formation, showed drastic changes in phosphorylation under hypoxia.

Conclusions

Phosphoproteomics of A. fumigatus revealed an involvement of autophagy under hypoxic growth conditions. Initial experiments have started to investigate the impact of autophagy on hypoxic growth in A. fumigatus