The monothiol glutaredoxin GrxD is essential for sensing iron starvation in Aspergillus fumigatus.

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.

The bZIP Transcription Factor HapX Is Post-Translationally Regulated to Control Iron Homeostasis in Aspergillus fumigatus.

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine161, sumoylation of lysine242 and phosphorylation of threonine319. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A. fumigatus.

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Constitutive activation of TORC1 signalling attenuates virulence in the cross-kingdom fungal pathogen Fusarium oxysporum.

The filamentous fungus Fusarium oxysporum causes vascular wilt disease in a wide range of plant species and opportunistic infections in humans. Previous work suggested that invasive growth in this pathogen is controlled by environmental cues such as pH and nutrient status. Here we investigated the role of Target Of Rapamycin Complex 1 (TORC1), a global regulator of eukaryotic cell growth and development. Inactivation of the negative regulator Tuberous Sclerosis Complex 2 (Tsc2), but not constitutive activation of the positive regulator Gtr1, in F. oxysporum resulted in inappropriate activation of TORC1 signalling under nutrient-limiting conditions. The tsc2Δ mutants showed reduced colony growth on minimal medium with different nitrogen sources and increased sensitivity to cell wall or high temperature stress. Furthermore, these mutants were impaired in invasive hyphal growth across cellophane membranes and exhibited a marked decrease in virulence, both on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, invasive hyphal growth in tsc2Δ strains was rescued by rapamycin-mediated inhibition of TORC1. Collectively, these results reveal a key role of TORC1 signalling in the development and pathogenicity of F. oxysporum and suggest new potential targets for controlling fungal infections.

Identification of virulence genes in Fusarium oxysporum f. sp. lycopersici by large-scale transposon tagging.

Forward genetic screens are efficient tools for the dissection of complex biological processes, such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici by inserting the novel modified impala element imp160::gfp upstream of the Aspergillus nidulans niaD gene, followed by transactivation with a constitutively expressed transposase. A collection of 2072 Nia(+) revertants was obtained from reporter strain T12 and screened for alterations in virulence, using a rapid assay for invasive growth on apple slices. Seven strains exhibited reduced virulence on both apple slices and intact tomato plants. Five of these were true revertants showing the re-insertion of imp160::gfp within or upstream of predicted coding regions, whereas the other two showed either excision without re-insertion or no excision. Linkage between imp160::gfp insertion and virulence phenotype was determined in four transposon-tagged loci using targeted deletion in the wild-type strain. Knockout mutants in one of the genes, FOXG_00016, displayed significantly reduced virulence, and complementation of the original revertant with the wild-type FOXG_00016 allele fully restored virulence. FOXG_00016 has homology to the velvet gene family of A. nidulans. The high rate of untagged virulence mutations in the T12 reporter strain appears to be associated with increased genetic instability, possibly as a result of the transactivation of endogenous transposable elements by the constitutively expressed transposase.