The regulation of zinc homeostasis by the ZafA transcriptional activator is essential for Aspergillus fumigatus virulence

Abstract

SummaryWe have previously shown that Aspergillus fumigatus is able to grow in zinc-limiting media and that this ability is regulated at transcriptional level by both the availability of zinc and pH. When A. fumigatus grows as a pathogen, it must necessarily obtain zinc from the zinc-limiting environment provided by host tissue. Accordingly, the regulation of zinc homeostasis by some zinc-responsive transcriptional regulator in A. fumigatus must be essential for fungal growth within tissues of an immunocompromised host and, in turn, for pathogenicity. Here we provide evidence of the role of the zafA gene in regulating zinc homeostasis and its relevance in the virulence of A. fumigatus. Thus, we observed that (i) zafA can functionally replace the ZAP1 gene from Saccharomyces cerevisiae that encodes the zinc-responsive transcriptional activator Zap1 protein; (ii) the expression of zafA itself is induced in zinc-limiting media and repressed by zinc; (iii) deletion of zafA impairs the germination and growth capacity of A. fumigatus in zinc-limiting media; and (iv) the deletion of zafA abrogates A. fumigatus virulence in a murine model of invasive aspergillosis. In light of these observations, we concluded that ZafA is a zinc-responsive transcriptional activator that represents an essential attribute for A. fumigatus pathogenicity. Consequently, ZafA may constitute a new target for the development of chemotherapeutic agents against Aspergillus, because no zafA orthologues have been found in mammals.

Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the zrfC and aspf2 Genes

“…In the za P region, there are three 15-bp consensus sequences with a common 5Ј-CAAGGT-3Ј core. These 15-bp sequences were designated ZR1 to ZR3 (for zinc response), since preliminary data obtained at our laboratory have indicated that they are cis-acting sequences required for regulating the expression of zrfC and aspf2 at the transcriptional level (unpublished data), most likely through binding of the zincresponsive transcriptional activator ZafA (28). In addition, in the za P region there are six PacC-like binding motifs (5Ј-GCC ARG-3Ј) (42) designated PR1 to PR6 (for pH response).…”

“…Expression of both zrfC and aspf2 is induced in zinc-limiting media by the ZafA zinc-responsive transcriptional activator. The ZafA protein of A. fumigatus activates the transcription of the zrfA and zrfB genes under acidic zinc-limiting conditions (28). Therefore, it would be expected that the transcription of these genes as well as of others related to zinc homeostasis in A. fumigatus would also be regulated by ZafA under alkaline zinc-limiting conditions.…”

“…Indeed, scrutiny of the A. fumigatus genome revealed that it actually has eight genes encoding proteins (ZrfA to ZrfH) of the ZIP (Zrt-like, Irt-like Protein) family of zinc transporters usually involved in zinc uptake from the environment (8). However, apart from the zrfA and zrfB genes, which encode the acid ZrfA-ZrfB zinc uptake system (45), only the promoter region of zrfC has cis-acting sequences for both the PacC and ZafA transcriptional regulators that respectively regulate pH and zinc homeostasis in Aspergillus species (28,31). In this work we demonstrate that zrfC does indeed encode a transporter devoted to obtaining zinc from alkaline zinc-limiting media.…”

“…1B) was also expressed in ZHY3. To prevent any misinterpretation of the data due to differences at the transcriptional level, each gene was expressed from a centromeric plasmid under the control of the ZRT1 promoter, as reported previously (28,45). The acidic zinc-limiting medium (pH 4.4) was supplemented with 1 mM EDTA (SDAEB4) to further limit zinc availability.…”

Aspergillus fumigatus: virulence genes in a street-smart mold

“…A. fumigatus uses siderophores for both iron acquisition and intracellular iron storage [51], and disrupting the siderophore biosynthetic pathway at multiple steps has revealed that A. fumigatus relies heavily on these low-molecular weight chelators for growth in the iron-limited environment of the host [51][52][53]. Zinc also has limited bioavailability in vivo, and the zincresponsive transcriptional activator ZafA is required to support virulence, presumably by enhancing zinc uptake mechanisms [54].…”