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

Abstract

Aspergillus fumigatus has three zinc transporter-encoding genes whose expression is regulated by both pH and the environmental concentration of zinc. We have previously reported that the zrfA and zrfB genes of A. fumigatus are transcribed at higher levels and are required for fungal growth under acidic zinc-limiting conditions whereas they are dispensable for growth in neutral or alkaline zinc-limiting media. Here we report that the transporter of the zinc uptake system that functions in A. fumigatus growing in neutral or alkaline environments is encoded by zrfC. The transcription of zrfC occurs divergently with respect to the adjacent aspf2 gene, which encodes an immunodominant antigen secreted by A. fumigatus. The two genes-zrfC and aspf2-are required to different extents for fungal growth in alkaline and extreme zinc-limiting media. Indeed, these environmental conditions induce the simultaneous transcription of both genes mediated by the transcriptional regulators ZafA and PacC. ZafA upregulates the expression of zrfC and aspf2 under zinc-limiting conditions regardless of the ambient pH, whereas PacC represses the expression of these genes under acidic growth conditions. Interestingly, the mode of action of PacC for zrfC-aspf2 transcription contrasts with the more widely accepted model for PacC function, according to which under alkaline growth conditions PacC would activate the transcription of alkaline-expressed genes but would repress the transcription of acid-expressed genes. In sum, this report provides a good framework for investigating several important aspects of the biology of species of Aspergillus, including the repression of alkaline genes by PacC at acidic pH and the interrelationship that must exist between tissue pH, metal availability in the host tissue, and fungal virulence.

Fig. 1.

Structural features of the za^P region and the ZrfC protein. (A) Schematic representation of the za^P region. The pH response sites (PR) and the putative zinc-response elements (ZR) are represented as gray- and black-shaded boxes, respectively. (B) Amino acid sequence of ZrfC. The predicted signal peptide and transmembrane domains are underlined and in bold lowercase, respectively. The four putative zinc-binding domains at the N terminus are shown separately for comparison. (C) Topological model of ZrfC insertion in the plasma membrane.

Fig. 2.

Analysis by Northern blotting of zrfA, zrfB, zrfC, and aspf2 transcription under both zinc-limiting and zinc-replete acidic and alkaline conditions. The AF14 strain was grown for 20 h at 37°C in acid SDA medium (pH 4.4) or alkaline SDN medium (pH 7.5) with a supplement of 100 μM Zn²⁺ (+) or without a supplement of Zn²⁺ (−), as indicated at the top of each lane.

Fig. 3.

Functional analysis of zrfC in the S. cerevisiae background. The yeast strain ZHY3 was transformed with derivative pRS316 plasmids carrying, under the control of the ZRT1 promoter, the coding sequence of ZRT1 (pMC5-HSET), zrfA (pZHA1), zrfB (pZHA2), zrfC (pZSF30), or zrfC^Δ13→622 (zrfC^ΔN) (pZSF310), as indicated on the left side of each picture. The DY1457 strain transformed with pRS316 is formally considered to be a wild type (wt). A total of 10⁴ yeast cells were spotted onto SDA zinc-limiting agar plates buffered at pH 4.4 (A) or 7.5 (B) with 100 mM potassium phosphate and supplemented with Zn²⁺ at the specified concentrations (0 to 2,000 μM). Acid plates were supplemented with 1 mM EDTA and incubated for 2 days at 28°C. Alkaline plates were supplemented with 0.1 mM EDTA and incubated for 2 and 4 days at 28°C.

Fig. 4.

Construction and phenotypic analysis of zrfCΔ strains. (A) A 1.86-kb NheI-XmnI DNA genomic fragment containing the complete coding sequence of zrfC in the CEA17 and AF15 strains (delimited with triangles) was replaced by the lacI-pyrG-lacI cassette (gray arrow flanked by dotted arrows) in the zrfCΔ (AF431) and zrfAΔzrfBΔzrfCΔ (AF251) strains by the use of a 7.22-kb DNA fragment (delimited with closed squares) obtained from plasmid pZRF35 as transforming DNA. The thinner arrows indicate putative open reading frames surrounding the zrfC gene. The pyrG gene of AF251 was removed by spontaneous intrachromosomal recombination to generate the uridine-uracil-auxotrophic strain AF2511, in which the zrfC coding sequence had been replaced by only one lacI fragment (dotted arrow delimited with triangles). All strains harbored the correct integration event at the zrfC locus, as verified by Southern blotting analyses, using as a probe a DNA fragment obtained by PCR with the oligonucleotide pair JA8 and JA26 and plasmid pZRF35 as the template. Only relevant restriction sites are indicated. The source of the genomic DNA, the restriction enzymes used in the digestions, and the sizes of the fragments detected that match the expected sizes are specifically indicated in each panel. (B) Growth of A. fumigatus strains AF431 (zrfCΔ), AF10 (zrfAΔzrfBΔ), and AF251 (zrfAΔzrfBΔzrfCΔ) on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn²⁺ or supplemented with 1 to 1,000 μM Zn²⁺, as indicated at the top of each panel.

Fig. 5.

Construction and phenotypic analyses of AF2511-derivative pyrG⁺ strains of A. fumigatus. (A) Construction of A. fumigatus strains that express zrfA (AF751), zrfB (AF761), zrfC (AF731), or zrfC^Δ13→622 (zrfC^ΔN; AF791) at the pyrG locus. All strains harbored the correct integration event, as verified by Southern blotting analyses (not shown). The dashed boxes represent the terminator of the aspnd1 gene, as described in Materials and Methods. (B) Growth of the same strains on both acid (SDAE, pH 4.5) and alkaline (SDNE, pH 7.5) zinc-limiting agar media not supplemented with Zn²⁺ or supplemented with 1 to 1,000 μM Zn²⁺, as indicated at the top of each panel.

Fig. 6.

Transcription analysis by Northern blotting of zrfC and aspf2 in the A. fumigatus zafAΔ null (AF17) and zafA⁺ revertant (AF56R) strains grown for 20 h at 37°C in the alkaline SDN medium with a supplement of 100 μM Zn²⁺ (+) or without a supplement of Zn²⁺ (−), as indicated at the top of each lane.

Fig. 7.

Transcription analysis by Northern blotting of zrfC and aspf2 in the pacC^c (AF58) and pacC^+/− (AF60) strains grown in either the acid SDA or alkaline SDN medium for 20 h at 37°C, both with a supplement of 100 μM Zn²⁺ (+) or without a supplement of Zn²⁺ (−), as indicated at the top of each lane.

Fig. 8.

Construction and phenotypic analysis of aspf2Δ strains. (A) A 1.2-kb BstZ17I-XhoI DNA genomic fragment containing the complete coding sequence of aspf2 in CEA17 (delimited with triangles) was replaced by the lacI-pyrG-lacI cassette (gray arrow flanked by dotted arrows) in the aspf2Δ (AF811) strain by the use of a 6.65-kb DNA fragment obtained from plasmid pASPF25 as transforming DNA. All strains harbored the correct integration event at the aspf2 locus, as verified by Southern blotting using as a probe a mixture of a DNA fragment obtained by PCR with the oligonucleotide pair JA187 and JA26 and plasmid pASPF25 as the template and a SmaI-BglII fragment obtained from plasmid pZRF39. Only relevant restriction sites are indicated. The source of the genomic DNA, the restriction enzymes used in the digestions, and the sizes of the fragments detected that match the expected sizes are specifically indicated in each panel. (B) Growth of A. fumigatus strains AF811 (aspf2Δ), AF881 (aspf2⁺), and AF431 (zrfCΔ) on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn²⁺ or supplemented with 1 to 100 μM Zn²⁺, as indicated at the top of each panel.

Fig. 9.

Influence of zrfC or aspf2 expression on the growth ability in acidic media of the AF801 (za^PR → zrfC) and AF891 (za^PR → aspf2) strains, respectively, and transcription of zrfC and aspf2 in both strains. (A) Growth of AF801 and AF891 on both acid (SDAE; pH 4.5) and alkaline (SDNE; pH 7.5) zinc-limiting agar media not supplemented with Zn²⁺ or supplemented with 1 or 10 μM Zn²⁺, as indicated at the top of each panel. (B) Analysis by Northern blotting of the transcription of zrfC and aspf2 under the control of the za^PR region in the AF801 and AF891 strains. These strains were constructed following the same strategy used previously to construct prototrophic pyrG⁺ strains at the pyrG1 locus of A. fumigatus but using a fragment contained in either the pZRF320 (for zrfC) or pASPF361 (for aspf2) plasmid as transforming DNA. The expression levels of zrfC in the AF891 strain and aspf2 in AF801 were analyzed as endogenous controls for the correct functioning of the wild-type za^P regions carried by these strains. Expression of zrfC in the AF731 strain and that of aspf2 in AF881 were also analyzed as additional controls, since these strains also express at the pyrG loci zrfC and aspf2, respectively, but under the control of a wild-type za^P region. The za^PR region that drives transcription of the tested gene is depicted on the right side of each blot. All fungal strains were grown in either the acid SDA or alkaline SDN medium for 20 h at 37°C with a supplement of 100 μM Zn²⁺ (+) or without a supplement of Zn²⁺ (−), as indicated at the top of each lane.