The Transcription Factor ZafA Regulates the Homeostatic and Adaptive Response to Zinc Starvation in Aspergillus fumigatus

Abstract

One of the most important features that enables Aspergillus fumigatus to grow within a susceptible individual and to cause disease is its ability to obtain Zn2+ ions from the extremely zinc-limited environment provided by host tissues. Zinc uptake from this source in A. fumigatus relies on ZIP transporters encoded by the zrfA, zrfB and zrfC genes. The expression of these genes is tightly regulated by the ZafA transcription factor that regulates zinc homeostasis and is essential for A. fumigatus virulence. We combined the use of microarrays, Electrophoretic Mobility Shift Assays (EMSA) analyses, DNase I footprinting assays and in silico tools to better understand the regulation of the homeostatic and adaptive response of A. fumigatus to zinc starvation. We found that under zinc-limiting conditions, ZafA functions mainly as a transcriptional activator through binding to a zinc response sequence located in the regulatory regions of its target genes, although it could also function as a repressor of a limited number of genes. In addition to genes involved in the homeostatic response to zinc deficiency, ZafA also influenced, either directly or indirectly, the expression of many other genes. It is remarkable that the expression of many genes involved in iron uptake and ergosterol biosynthesis is strongly reduced under zinc starvation, even though only the expression of some of these genes appeared to be influenced directly or indirectly by ZafA. In addition, it appears to exist in A. fumigatus a zinc/iron cross-homeostatic network to allow the adaptation of the fungus to grow in media containing unbalanced Zn:Fe ratios. The adaptive response to oxidative stress typically linked to zinc starvation was also mediated by ZafA, as was the strong induction of genes involved in gliotoxin biosynthesis and self-protection against endogenous gliotoxin. This study has expanded our knowledge about the regulatory and metabolic changes displayed by A. fumigatus in response to zinc starvation and has helped us to pinpoint new ZafA target genes that could be important for fungal pathogens to survive and grow within host tissues and, hence, for virulence.

The interplay between zinc and iron homeostasis in Aspergillus fumigatus under zinc‐replete conditions relies on the iron‐mediated regulation of alternative transcription units of zafA and the basal amount of the ZafA zinc‐responsiveness transcription factor

“…Similarly, the expression of the zrfA , zrfB and zrfC was up‐regulated, whereas that of zafA , zrfF , zrcA and zrcC was down‐regulated compared to a Δ srbB mutant (Chung et al, ). Unlike all previous studies carried out under zinc‐replete conditions, we have recently shown that expression of hapX , srbA and srbB and that of several genes encoding proteins involved in iron‐uptake and ergosterol biosynthesis was down‐regulated in zinc‐limiting media under iron‐replete conditions (Vicentefranqueira et al, ). In summary, all previous findings strongly suggested the existence of an interconnection between the regulation of iron and zinc homeostasis, which caused us to contemplate how iron availability influences zafA expression and/or modulated ZafA function even under zinc‐replete conditions.…”

“…Actually, the expression of genes involved in iron uptake is down‐regulated in A. fumigatus as an adaptive response to growth in zinc‐limiting, iron‐replete media. This is most likely a mechanism to prevent iron toxicity under zinc starvation (Vicentefranqueira et al, ). In contrast, fungal growth in zinc‐replete media (10‐100 μM zinc) is strongly conditioned by iron, such that fungal growth decreases when the Zn:Fe ratio of the media becomes >3.2 due to iron shortage (Fig.…”

“…The transcription regulatory activity of ZafA and HapX appears to be modulated by the intracellular Zn:Fe ratio It was previously shown that the expression of genes hapX and srbA, which encode factors involved in the regulation of iron homeostasis, was down-regulated under zinc-limiting, iron-replete conditions (Vicentefranqueira et al, 2018). To investigate whether an excess of zinc influenced the expression and/or function of these regulators in addition to that of ZafA, we created a fungal strain (AF1059) that expressed zrfC in zinc-replete media under the control of the promoter of the actA gene from A. nidulans [PactA !…”

“…The ability to obtain zinc from host tissues is one of the most important biological traits that enables A. fumigatus to grow and cause disease in susceptible individuals (Amich et al, ; Amich and Calera, ). The regulation of the homeostatic and adaptive response to zinc starvation in A. fumigatus is mediated by the ZafA transcription factor (Vicentefranqueira et al, ), which is essential for fungal virulence (Moreno et al ., b). ZafA regulates gene expression through binding to the zinc responsive (ZR) motifs present in the regulatory regions of its target genes (Vicentefranqueira et al, ), as does the Zap1 transcription factor of Saccharomyces cerevisiae , which is the best characterized ZafA orthologue to date (Zhao and Eide, ).…”

“…The regulation of the homeostatic and adaptive response to zinc starvation in A. fumigatus is mediated by the ZafA transcription factor (Vicentefranqueira et al, 2018), which is essential for fungal virulence (Moreno et al, 2007b). ZafA regulates gene expression through binding to the zinc responsive (ZR) motifs present in the regulatory regions of its target genes (Vicentefranqueira et al, 2018), as does the Zap1 transcription factor of Saccharomyces cerevisiae, which is the best characterized ZafA orthologue to date (Zhao and Eide, 1997). The Zap1 regulon and the genes targeted directly by Zap1 have been well identified, and the mechanism of action of Zap1 has been studied in depth (Eide, 2009).…”