Alternative mechanisms of protection of Aspergillus fumigatus conidia against Reactive Oxygen Species

Objective: Aspergillus fumigatus is the main causative agent of invasive aspergillosis. Upon inhalation conidia pass the mucosal barrier and end up in the lung cavities, the alveoli. Surveilling lung macrophages and type II lung epithelial cells produce amongst others intra-and extracellular reactive oxygen and nitrogen species (ROS/RNS) to prevent fungal growth. ROS consists of superoxides, hydrogen peroxide and hydroxyl radicals. Fungal components involved in protection against these compounds are superoxide dismutase (O2 -), catalases (H2O2 ), L-DOPA melanine (OH. ) due to the existence of different oxidative states of the quinone residues. A. fumigatus produces DHN-melanine present in spores whereas pyomelanine is produced by hyphae. Both types of melanines were suggested to play a role in protection against ROS but the evidence of their protective role is very thin. In addition, how these compounds protect at a chemical level has not been investigated. Our objective is to unravel the protective mechanisms of fungal components against ROS and RNS. We expect that surface exposed and/or secreted fungal component are involved in protection. In this study we have investigated the sensitivity of spores of WT A. fumigatus strain and mutants with disruption in genes involved in production of DHN-melanine (PksP), pyomelanine (HppD) or the rodlets (Roda or RodB) for hydrogen peroxide and hydroxyl radicals. Methods: Spores or fungal cells were incubated with different concentrations of H2O2 or hydroxyl radicals for several time intervals and survival was determined after dilution plating on PDA agar media and counting CFU. Spores were plated on minimal medium plates containing different concentrations of H2O2 and zones of inhibition were determined. L-tyrosine was added to culture media to determine the role of pyomelanine. Results: Comparison of sensitivity for hydrogen peroxide or hydroxyl radicals of the WT A. fumigatus strain with any of the mutants in different types of assays did not reveal any change in sensitivity for the tested ROS. In contrast, C. neoformans grown in the presence of L-DOPA and producing L-DOPA melanine was found to be protected against hydroxyl radicals. Conclusion: The rodlet and the melanin layer present at the outside of the conidia of A. fumigatus does not play a role in the protection against hydrogen peroxide and hydroxyl radicals, this in contrast to the role of L-DOPA-malanine in the cell wall of C. neoformans. The resistance of fumigatus spores to hydroxyl radicals is very striking and we propose that spores of A. fumigatus have a novel protective mechanism against this component and a possible role of secondary metabolites is discussed.