At the metal-metabolite interface in Aspergillus fumigatus: towards untangling the intersecting roles of zinc and gliotoxin.

Cryptic links between apparently unrelated metabolic systems represent potential new drug targets in fungi. Evidence of such a link between zinc and gliotoxin (GT) biosynthesis in Aspergillus fumigatus is emerging. Expression of some genes of the GT biosynthetic gene cluster gli is influenced by the zinc-dependent transcription activator ZafA, zinc may relieve GT-mediated fungal growth inhibition and, surprisingly, GT biosynthesis is influenced by zinc availability. In A. fumigatus, dithiol gliotoxin (DTG), which has zinc-chelating properties, is converted to either GT or bis-dethiobis(methylthio)gliotoxin (BmGT) by oxidoreductase GliT and methyltransferase GtmA, respectively. A double deletion mutant lacking both GliT and GtmA was previously observed to be hypersensitive to exogenous GT exposure. Here we show that compared to wild-type exposure, exogenous GT and the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibit A. fumigatus ΔgliTΔgtmA growth, specifically under zinc-limiting conditions, which can be reversed by zinc addition. While GT biosynthesis is evident in zinc-depleted medium, addition of zinc (1 µM) suppressed GT and activated BmGT production. In addition, secretion of the unferrated siderophore, triacetylfusarinine C (TAFC), was evident by A. fumigatus wild-type (at >5 µM zinc) and ΔgtmA (at >1 µM zinc) in a low-iron medium. TAFC secretion suggests that differential zinc-sensing between both strains may influence fungal Fe3+ requirement. Label-free quantitative proteomic analysis of both strains under equivalent differential zinc conditions revealed protein abundance alterations in accordance with altered metabolomic observations, in addition to increased GliT abundance in ΔgtmA at 5 µM zinc, compared to wild-type, supporting a zinc-sensing deficiency in the mutant strain. The relative abundance of a range of oxidoreductase- and secondary metabolism-related enzymes was also evident in a zinc- and strain-dependent manner. Overall, we elaborate new linkages between zinc availability, natural product biosynthesis and oxidative stress homeostasis in A. fumigatus.

Repression of the acid ZrfA/ZrfB zinc-uptake system of Aspergillus fumigatus mediated by PacC under neutral, zinc-limiting conditions.

The ZrfA and ZrfB transporters are components of a zinc-uptake system of Aspergillus fumigatus that mainly operates under acidic, zinc-limiting conditions. Expression of the genes zrfA and zrfB is up-regulated by the transcriptional activator ZafA in both acidic and neutral, zinc-limiting media. The transcription of zafA is not influenced by PacC, which is the transcriptional regulator involved in regulating pH homeostasis in Aspergillus. However, at neutral pH the expression of both zrfA and zrfB is significantly reduced. In this work, the repression of zrfA and zrfB in neutral and alkaline, zinc-limiting media was found to be mediated by the transcriptional regulator PacC.

Involvement of Sulfur in the Biosynthesis of Essential Metabolites in Pathogenic Fungi of Animals, Particularly Aspergillus spp.: Molecular and Therapeutic Implications.

Fungal sulfur uptake is required for incorporation into the sidechains of the amino acids cysteine and methionine, and is also essential for the biosynthesis of the antioxidant glutathione (GSH), S-adenosylmethionine (SAM), the key source of methyl groups in cellular transmethylation reactions, and S-adenosylhomocysteine (SAH). Biosynthesis of redox-active gliotoxin in the opportunistic fungal pathogen Aspergillus fumigatus has been elucidated over the past 10 years. Some fungi which produce gliotoxin-like molecular species have undergone unexpected molecular rewiring to accommodate this high-risk biosynthetic process. Specific disruption of gliotoxin biosynthesis, via deletion of gliK, which encodes a γ-glutamyl cyclotransferase, leads to elevated intracellular antioxidant, ergothioneine (EGT), levels, and confirms crosstalk between the biosynthesis of both sulfur-containing moieties. Gliotoxin is ultimately formed by gliotoxin oxidoreductase GliT-mediated oxidation of dithiol gliotoxin (DTG). In fact, DTG is a substrate for both GliT and a bis-thiomethyltransferase, GtmA. GtmA converts DTG to bisdethiobis(methylthio)gliotoxin (BmGT), using 2 mol SAM and resultant SAH must be re-converted to SAM via the action of the Methyl/Met cycle. In the absence of GliT, DTG fluxes via GtmA to BmGT, which results in both SAM depletion and SAH overproduction. Thus, the negative regulation of gliotoxin biosynthesis via GtmA must be counter-balanced by GliT activity to avoid Methyl/Met cycle dysregulation, SAM depletion and trans consequences on global cellular biochemistry in A. fumigatus. DTG also possesses potent Zn2+ chelation properties which positions this sulfur-containing metabolite as a putative component of the Zn2+ homeostasis system within fungi. EGT plays an essential role in high-level redox homeostasis and its presence requires significant consideration in future oxidative stress studies in pathogenic filamentous fungi. In certain filamentous fungi, sulfur is additionally indirectly required for the formation of EGT and the disulfide-bridge containing non-ribosomal peptide, gliotoxin, and related epipolythiodioxopiperazines. Ultimately, interference with emerging sulfur metabolite functionality may represent a new strategy for antifungal drug development.

Targeting zinc homeostasis to combat Aspergillus fumigatus infections.

Aspergillus fumigatus is able to invade and grow in the lungs of immunosuppressed individuals and causes invasive pulmonary aspergillosis. The concentration of free zinc in living tissues is much lower than that required for optimal fungal growth in vitro because most of it is tightly bound to proteins. To obtain efficiently zinc from a living host A. fumigatus uses the zinc transporters ZrfA, ZrfB, and ZrfC. The ZafA transcriptional regulator induces the expression of all these transporters and is essential for virulence. Thus, ZafA could be targeted therapeutically to inhibit fungal growth. The ZrfC transporter plays the major role in zinc acquisition from the host whereas ZrfA and ZrfB rather have a supplementary role to that of ZrfC. In addition, only ZrfC enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mn-chelating protein synthesized and released by neutrophils within the fungal abscesses of immunosuppressed non-leucopenic animals. Hence, fungal survival in these animals would be undermined upon blocking therapeutically the function of ZrfC. Therefore, both ZafA and ZrfC have emerged as promising targets for the discovery of new antifungals to treat Aspergillus infections.

Culture conditions for zinc- and pH-regulated gene expression studies in Aspergillus fumigatus.

In Aspergillus fumigatus, the regulation of zinc homeostasis is strongly influenced by environmental pH. Thus, the study of zinc-regulated gene expression in A. fumigatus requires controlling variations in culture pH, as this may affect zinc availability. However, depending on the nitrogen source, the pH of the culture can change dramatically over time. In addition, due to the ubiquitous distribution of zinc and that it is an essential micronutrient required in minute amounts for optimal fungal growth, neither buffering of the culture media to prevent pH variations nor the use of chelating agents is advisable if mycelium is to be used for expression analyses. In this work, the growth of A. fumigatus in several culture media was examined in order to determine the conditions yielding mycelia suitable for gene expression analyses in acid and neutral media, regardless of zinc availability. Our results showed that a zinc-limiting synthetic basal medium could be readily converted into a zinc-replete one and subsequently into acid or neutral medium by using, respectively, ammonium or nitrate as nitrogen source.

Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynthesis and adaptation to oxidative stress.

Ssk1p of Candida albicans is a putative response regulator protein of the Hog1 two-component signal transduction system. In Saccharomyces cerevisiae, the phosphorylation state of Ssk1p determines whether genes that promote the adaptation of cells to osmotic stress are activated. We have previously shown that C. albicans SSK1 does not complement the ssk1 mutant of S. cerevisiae and that the ssk1 mutant of C. albicans is not sensitive to sorbitol. In this study, we show that the C. albicans ssk1 mutant is sensitive to several oxidants, including hydrogen peroxide, t-butyl hydroperoxide, menadione, and potassium superoxide when each is incorporated in yeast extract-peptone-dextrose (YPD) agar medium. We used DNA microarrays to identify genes whose regulation is affected by the ssk1 mutation. RNA from mutant cells (strain CSSK21) grown in YPD medium for 3 h at 30 degrees C was reverse transcribed and then compared with similarly prepared RNA from wild-type cells (CAF2). We observed seven genes from mutant cells that were consistently up regulated (three-fold or greater compared to CAF2). In S. cerevisiae, three (AHP1, HSP12, and PYC2) of the seven genes that were up regulated provide cells with an adaptation function in response to oxidative stress; another gene (GPH1) is regulated under stress conditions by Hog1p. Three other genes that are up regulated encode a cell surface protein (FLO1), a mannosyl transferase (MNN4-4), and a putative two-component histidine kinase (CHK1) that regulates cell wall biosynthesis in C. albicans. Of the down-regulated genes, ALS1 is a known cell adhesin in C. albicans. Verification of the microarray data was obtained by reverse transcription-PCR for HSP12, AHP1, CHK1, PYC2, GPH1, ALS1, MNN4-4, and FLO1. To further determine the function of Ssk1p in the Hog1p signal transduction pathway in C. albicans, we used Western blot analysis to measure phosphorylation of Hog1p in the ssk1 mutant of C. albicans when grown under either osmotic or oxidative stress. We observed that Hog1p was phosphorylated in the ssk1 mutant of C. albicans when grown in a hyperosmotic medium but was not phosphorylated in the ssk1 mutant when the latter was grown in the presence of hydrogen peroxide. These data indicate that C. albicans utilizes the Ssk1p response regulator protein to adapt cells to oxidative stress, while its role in the adaptation to osmotic stress is less certain. Further, SSK1 appears to have a regulatory function in some aspects of cell wall biosynthesis. Thus, the functions of C. albicans SSK1 differ from those of S. cerevisiae SSK1.

Repression of the acid ZrfA/ZrfB zinc-uptake system of Aspergillus fumigatus mediated by PacC under neutral, zinc-limiting conditions

The ZrfA and ZrfB transporters are components of a zinc-uptake system of Aspergillus fumigatus that mainly operates under acidic, zinc-limiting conditions. Expression of the genes zrfA and zrfB is up-regulated by the transcriptional activator ZafA in both acidic and neutral, zinc-limiting media. The transcription of zafA is not influenced by PacC, which is the transcriptional regulator involved in regulating pH homeostasis in Aspergillus. However, at neutral pH the expression of both zrfA and zrfB is significantly reduced. In this work, the repression of zrfA and zrfB in neutral and alkaline, zinc-limiting media was found to be mediated by the transcriptional regulator PacC (AU)

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Culture conditions for zinc- and pH-regulated gene expression studies in Aspergillus fumigatus

In Aspergillus fumigatus, the regulation of zinc homeostasis is strongly influenced by environmental pH. Thus, the study of zinc-regulated gene expression in A. fumigatus requires controlling variations in culture pH, as this may affect zinc availability. However, depending on the nitrogen source, the pH of the culture can change dramatically over time. In addition, due to the ubiquitous distribution of zinc and that it is an essential micronutrient required in minute amounts for optimal fungal growth, neither buffering of the culture media to prevent pH variations nor the use of chelating agents is advisable if mycelium is to be used for expression analyses. In this work, the growth of A. fumigatus in several culture media was examined in order to determine the conditions yielding mycelia suitable for gene expression analyses in acid and neutral media, regardless of zinc availability. Our results showed that a zinc-limiting synthetic basal medium could be readily converted into a zinc-replete one and subsequently into acid or neutral medium by using, respectively, ammonium or nitrate as nitrogen source (AU)

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