Phospho- and redox proteomic analysis of the A. fumigatus response to hypoxic stress

The filamentous fungus 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. The transcription factor SrbA is the central regulator of the fungal hypoxia response. However, little is known about the adaptation to hypoxia on the protein and posttranslational level. Therefore, we performed quantitative proteomics, including phospho- and redox proteomic analyses by comparing fungal mycelium grown under either normoxic (21% O2) or hypoxic (0.2% O2) conditions in an oxygen-controlled fermenter. The goal was to find novel regulatory circuits in hypoxic adaptation. 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. For the identification of oxidative thiol modifications, we applied the thiol trapping OxICAT technique. 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 precursor ion quantification. We quantified in total 4607 proteins, of which 407 proteins and 1391 phosphopeptides showed significantly different abundance upon hypoxia (fold change >2, 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 further that the mitotic cell cycle and autophagy processes are differentially regulated under hypoxic growth. The OxICAT method revealed 44 cysteine-containing peptides with a decreased level of thiol oxidation and 36 peptides with an increased level of thiol oxidation under hypoxic growth. A cysteine in the osmotic stress regulating mitogen-activated protein kinase Hog1/SakA exhibited a particularly drastic level of thiol oxidation under hypoxic growth. Based on these findings the identified regulatory mechanisms will be investigated further in A. fumigatus on the genetic level.