Redox Proteomic Analysis Reveals Oxidative Modifications of Proteins by Increased Levels of Intracellular Reactive Oxygen Species during Hypoxia Adaptation of Aspergillus fumigatus.

Aspergillus fumigatus faces abrupt changes in oxygen concentrations at the site of infection. An increasing number of studies has demonstrated that elevated production of intracellular reactive oxygen species (ROS) under low oxygen conditions plays a regulatory role in modulating cellular responses for adaptation to hypoxia. To learn more about this process in A. fumigatus, intracellular ROS production during hypoxia has been determined. The results confirm increased amounts of intracellular ROS in A. fumigatus exposed to decreased oxygen levels. Moreover, nuclear accumulation of the major oxidative stress regulator AfYap1 is observed after low oxygen cultivation. For further analysis, iodoTMT labeling of redox-sensitive cysteine residues is applied to identify proteins that are reversibly oxidized. This analysis reveals that proteins with important roles in maintaining redox balance and protein folding, such as the thioredoxin Asp f 29 and the disulfide-isomerase PdiA, undergo substantial thiol modification under hypoxia. The data also show that the mitochondrial respiratory complex IV assembly protein Coa6 is significantly oxidized by hypoxic ROS. Deletion of the corresponding gene results in a complete absence of hypoxic growth, indicating the importance of complex IV during adaptation of A. fumigatus to oxygen-limiting conditions.

Functional analysis of the Aspergillus fumigatus kinome identifies a druggable DYRK kinase that regulates septal plugging.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.