Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence

Abstract

Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of ΔfhpA, ΔfhpB, or ΔgnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.

Identification and Characterization of a Novel Aspergillus fumigatus Rhomboid Family Putative Protease, RbdA, Involved in Hypoxia Sensing and Virulence

“…4). In contrast, regulation of the flavohemoprotein gene fhpA, whose transcription was found to be commonly responsive to NO and hypoxia (8,30), did not depend on either SrbA or RbdA. These results suggest that RbdA is tightly connected to the transcriptional activator function of SrbA.…”

Insights into the cellular responses to hypoxia in filamentous fungi

“…Flavohemoglobins, like FhpA have NO dioxygenase activity and are generally regarded as part of an NO detoxification system catalyzing the NADPH-dependant oxidation of toxic NO to NO 3 − (Gardner et al 1998). Respective null mutants showed indeed NO-sensitive phenotypes in A. nidulans and A. fumigatus (Schinko et al 2010;Lapp et al 2014). However, NO levels generated during hypoxia might not cause detrimental cellular damage, as at least in A. fumigatus, an fhpA deletion mutant did not display any visible growth defect in hypoxic atmospheres .…”

Riboflavin and pantothenic acid biosynthesis are crucial for iron homeostasis and virulence in the pathogenic mold Aspergillus fumigatus

“…We confirmed here the importance of riboflavin for NO-detoxification and siderophore biosynthesis in A. fumigatus . In contrast to NO-detoxification, which appears to be dispensable for virulence of A. fumigatus [41], siderophore biosynthesis has been shown to be essential for virulence of several animal and plant pathogenic fungi [42]. Similarly, pantothenic acid is of central importance for primary metabolism as it is essential for coenzyme A biosynthesis and consequently for fatty acid metabolism, carbohydrate metabolism and tricarboxylic acid cycle [43].…”