Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

Regulation of zinc homeostatic genes by environmental pH in the filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus can grow over a broad range of pH values even though zinc availability is greatly conditioned by ambient pH. It has been previously shown that regulation of zinc homeostatic genes in this fungus relies on the transcription factor ZafA. In addition, their expression is further modulated by the transcription factor PacC depending on ambient pH, which allows this fungus to grow in diverse types of niches, including soils and the lungs of immunosuppressed hosts. In this work the regulation by PacC of genes zrfB and zrfC that are expressed, respectively, under acidic and alkaline zinc-limiting conditions have been analysed in detail. Thus, data that extend the current model for PacC function, including the role of the full-length PacC72 protein and the PacC processed forms (PacC53 and PacC27 ) on gene expression has been provided, and a new mechanism for the repression of acid-expressed genes in alkaline media based on interference with the start of transcription has been described. Moreover, it was proposed that the transcription of both acid-expressed and alkaline-expressed genes under zinc-limiting conditions might also rely on a third factor (putatively Pontin/Reptin), which may be required to integrate the action of PacC and ZafA into gene specific transcriptional responses.

Point Mutation or Overexpression of Aspergillus fumigatus cyp51B, Encoding Lanosterol 14α-Sterol Demethylase, Leads to Triazole Resistance.

Aspergillus fumigatus is the most common cause of invasive fungal mold infections in immunocompromised individuals. Current antifungal treatment relies heavily on the triazole antifungals which inhibit fungal Erg11/Cyp51 activity and subsequent ergosterol biosynthesis. However, resistance, due primarily to cyp51 mutation, is rapidly increasing. A. fumigatus contains two Cyp51 isoenzymes, Cyp51A and Cyp51B. Overexpression and mutation of Cyp51A is a major cause of triazole resistance in A. fumigatus. The role of Cyp51B in generating resistance is unclear. Here, we show that overexpression or mutation of cyp51B results in triazole resistance. We demonstrate that introduction of a G457S Cyp51B mutation identified in a resistant clinical isolate results in voriconazole resistance in a naive recipient strain. Our results indicate that mutations in cyp51B resulting in clinical resistance do exist and should be monitored.

The novel Aspergillus fumigatus MAT1-2-4 mating-type gene is required for mating and cleistothecia formation.

Sexual propagation accompanied by recombination and the formation of spore-containing fruiting bodies is a cornerstone of fungal genetics and biology. In the human pathogen Aspergillus fumigatus sexual identity has previously been shown to be determined by MAT1-1-1 or MAT1-2-1 genes which act as transcriptional regulators and are present within idiomorphs found at the MAT locus. We here report the identification and first characterization of a further novel gene, termed MAT1-2-4, that is present in the MAT1-2 idiomorph of A. fumigatus. A mating-type swapping strategy was used to achieve an unbiased deletion of the MAT1-2-4 gene with no impact on MAT1-2-1 gene expression. Phenotypical characterization of the resulting strain revealed an inability to mate with the compatible MAT1-1 progenitor, demonstrating that the MAT1-2-4 gene product is a genuine mating-type factor required for correct sexual development. A GPI-anchored protein of unknown function was identified as interaction partner. However, no functional role in the mating process or ascosporogenesis could be demonstrated by deletion analysis for this latter protein, although a role in heterokaryon formation is suggested. Bioinformatic analysis also demonstrated the presence of MAT1-2-4 homologues in some, but not all, other Aspergillus species and the evolutionary origins and implications of the MAT1-2-4 gene are discussed.

Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant.

Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements ofA. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection.

Regulation of sulphur assimilation is essential for virulence and affects iron homeostasis of the human-pathogenic mould Aspergillus fumigatus.

Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.

The ZrfC alkaline zinc transporter is required for Aspergillus fumigatus virulence and its growth in the presence of the Zn/Mn-chelating protein calprotectin.

Aspergillus fumigatus can invade the lungs of immunocompromised individuals causing a life-threatening disease called invasive pulmonary aspergillosis (IPA). To grow in the lungs, A. fumigatus obtains from the host all nutrients, including zinc. In living tissues, however, most zinc is tightly bound to zinc-binding proteins. Moreover, during infection the bioavailability of zinc can be further decreased by calprotectin, an antimicrobial Zn/Mn-chelating protein that is released by neutrophils in abscesses. Nevertheless, A. fumigatus manages to uptake zinc from and grow within the lungs of susceptible individuals. Thus, in this study we investigated the role of the zrfA, zrfB and zrfC genes, encoding plasma membrane zinc transporters, in A. fumigatus virulence. We showed that zrfC is essential for virulence in the absence of zrfA and zrfB, which contribute to fungal pathogenesis to a lesser extent than zrfC and are dispensable for virulence in the presence of zrfC. The special ability of ZrfC to scavenge and uptake zinc efficiently from lungtissue depended on its N-terminus, which is absent in the ZrfA and ZrfB transporters. In addition, under Zn- and/or Mn-limiting conditions zrfC enables A. fumigatus to grow in the presence of calprotectin, which is detected in fungal abscesses of non-leucopenic animals. This study extends our knowledge about the pathobiology of A. fumigatus and suggests that fungal zinc uptake could be a promising target for new antifungals.

Zinc acquisition: a key aspect in Aspergillus fumigatus virulence.

Zinc is an essential micronutrient required for the growth of all microorganisms. To grow in the lungs of a susceptible patient Aspergillus fumigatus must obtain zinc from the surrounding tissues. The concentration of Zn(2+) ions in living tissues is much lower than that required for optimal fungal growth in vitro because most of them are tightly bound to proteins at the physiological pH. However, A. fumigatus has several zinc transporters (ZrfA, ZrfB and ZrfC) that enable it to uptake zinc efficiently under the extreme zinc-limiting conditions provided by a susceptible host. The ZafA transcriptional regulator induces the expression of these transporters and is essential for virulence. ZrfC is required for fungal growth within the host tissues, whereas ZrfA and ZrfB play an accessory role. The zinc-scavenging capacity of ZrfC relies on its unusually long N-terminus. In addition, ZrfC also enables A. fumigatus to overcome the inhibitory effect of calprotectin, which is an antimicrobial Zn/Mn-chelating protein synthesized in high amounts by neutrophils, even in immunosuppressed non-leucopenic animals. In summary, the regulation of zinc homeostasis and zinc acquisition could be promising targets for the discovery and development of a new generation of antifungals for the treatment of invasive pulmonary aspergillosis.

The many roles of sulfur in the fungal-host interaction.

Sulfur is an essential macronutrient for life, and consequently, all living organisms must acquire it from external sources to thrive and grow. Sulfur is a constituent of a multitude of crucial molecules, such as the S-containing proteinogenic amino acids cysteine and methionine; cofactors and prosthetic groups, such as coenzyme-A and iron-sulfur (Fe-S) clusters; and other essential organic molecules, such as glutathione or S-adenosylmethionine. Additionally, sulfur in cysteine thiols is an active redox group that plays paramount roles in protein stability, enzyme catalysis, and redox homeostasis. Furthermore, H2S is gaining more attention as a crucial signaling molecule that influences metabolism and physiological functions. Given its importance, it is not surprising that sulfur plays key roles in the host-pathogen interaction. However, in contrast to its well-recognized involvement in the plant-pathogen interaction, the specific contributions of sulfur to the human-fungal interaction are much less understood. In this short review, I highlight some of the most important known mechanisms and propose directions for further research.

Importance of the Aspergillus fumigatus Mismatch Repair Protein Msh6 in Antifungal Resistance Development.

One of the systems responsible for the recognition and repair of mistakes occurring during cell replication is the DNA mismatch repair (MMR) system. Two major protein complexes constitute the MMR pathway: MutS and MutL. Here, we investigated the possible relation of four A. fumigatus MMR genes (msh2, msh6, pms1, and mlh1) with the development of azole resistance related to the phenomenon of multi-drug resistance. We examined the MMR gene variations in 163 Aspergillus fumigatus genomes. Our analysis showed that genes msh2, pms1, and mlh1 have low genetic variability and do not seem to correlate with drug resistance. In contrast, there is a nonsynonymous mutation (G240A) in the msh6 gene that is harbored by 42% of the strains, most of them also harboring the TR34/L98H azole resistance mechanism in cyp51A. The msh6 gene was deleted in the akuBKU80A. fumigatus strain, and the ∆msh6 isolates were analyzed for fitness, azole susceptibility, and virulence capacity, showing no differences compared with the akuBKU80 parental strain. Wild-type msh6 and Δmsh6 strains were grown on high concentrations of azole and other non-azole fungicides used in crop protection. A 10- and 2-fold higher mutation frequency in genes that confer resistance to boscalid and benomyl, respectively, were observed in Δmsh6 strains compared to the wild-type. This study suggests a link between Msh6 and fungicide resistance acquisition.

Functional analysis of the Aspergillus fumigatus kinome identifies a druggable DYRK kinase that regulates septal plugging.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

The role of methionine synthases in fungal metabolism and virulence.

Methionine synthases (MetH) catalyse the methylation of homocysteine (Hcy) with 5-methyl-tetrahydrofolate (5, methyl-THF) acting as methyl donor, to form methionine (Met) and tetrahydrofolate (THF). This function is performed by two unrelated classes of enzymes that differ significantly in both their structures and mechanisms of action. The genomes of plants and many fungi exclusively encode cobalamin-independent enzymes (EC.2.1.1.14), while some fungi also possess proteins from the cobalamin-dependent (EC.2.1.1.13) family utilised by humans. Methionine synthase's function connects the methionine and folate cycles, making it a crucial node in primary metabolism, with impacts on important cellular processes such as anabolism, growth and synthesis of proteins, polyamines, nucleotides and lipids. As a result, MetHs are vital for the viability or virulence of numerous prominent human and plant pathogenic fungi and have been proposed as promising broad-spectrum antifungal drug targets. This review provides a summary of the relevance of methionine synthases to fungal metabolism, their potential as antifungal drug targets and insights into the structures of both classes of MetH.

Duox is the primary NADPH oxidase responsible for ROS production during adult caudal fin regeneration in zebrafish.

Sustained elevated levels of reactive oxygen species (ROS) have been shown to be essential for regeneration in many organisms. This has been shown primarily via the use of pharmacological inhibitors targeting the family of NADPH oxidases (NOXes). To identify the specific NOXes involved in ROS production during adult caudal fin regeneration in zebrafish, we generated nox mutants for duox, nox5 and cyba (a key subunit of NOXes 1-4) and crossed these lines with a transgenic line ubiquitously expressing HyPer, which permits the measurement of ROS levels. Homozygous duox mutants had the greatest effect on ROS levels and rate of fin regeneration among the single mutants. However, duox:cyba double mutants showed a greater effect on fin regeneration than the single duox mutants, suggesting that Nox1-4 also play a role during regeneration. This work also serendipitously found that ROS levels in amputated adult zebrafish fins oscillate with a circadian rhythm.

Potential Implication of Azole Persistence in the Treatment Failure of Two Haematological Patients Infected with Aspergillus fumigatus.

Invasive aspergillosis (IA) is a major cause of morbidity and mortality in patients receiving allogeneic haematopoieticcell transplantation. The deep immunosuppression and a variety of potential additional complications developed in these patients result in IA reaching mortality rates of around 50-60%. This mortality is even higher when the patients are infected with azole-resistant isolates, demonstrating that, despite the complexity of management, adequate azole treatment can have a beneficial effect. It is therefore paramount to understand the reasons why antifungal treatment of IA infections caused by azole-susceptible isolates is often unsuccessful. In this respect, there are already various factors known to be important for treatment efficacy, for instance the drug concentrations achieved in the blood, which are thus often monitored. We hypothesize that antifungal persistence may be another important factor to consider. In this study we present two case reports of haematological patients who developed proven IA and suffered treatment failure, despite having been infected with susceptible isolates, receiving correct antifungal treatment and reaching therapeutic levels of the azole. Microbiological analysis of the recovered infective isolates showed that the patients were infected with multiple strains, several of which were persisters to voriconazole and/or isavuconazole. Therefore, we propose that azole persistence may have contributed to therapeutic failure in these patients and that this phenomenon should be considered in future studies.

Functional analysis of the Aspergillus fumigatus kinome reveals a DYRK kinase involved in septal plugging is a novel antifungal drug target.

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. The azole class of antifungals represent first line therapeutics for most of these infections however resistance is rising. Identification of novel antifungal targets that, when inhibited, synergise with the azoles will aid the development of agents that can improve therapeutic outcomes and supress the emergence of resistance. As part of the A. fumigatus genome-wide knockout program (COFUN), we have completed the generation of a library that consists of 120 genetically barcoded null mutants in genes that encode the protein kinase cohort of A. fumigatus. We have employed a competitive fitness profiling approach (Bar-Seq), to identify targets which when deleted result in hypersensitivity to the azoles and fitness defects in a murine host. The most promising candidate from our screen is a previously uncharacterised DYRK kinase orthologous to Yak1 of Candida albicans, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. Here we show that the orthologue YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress via phosphorylation of the Woronin body tethering protein Lah. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and impacts growth in murine lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit Yak1 in C. albicans prevents stress mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

Aspergillus fumigatus Can Display Persistence to the Fungicidal Drug Voriconazole.

Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first-line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non-mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCE The phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized the response of the prominent fungal pathogen Aspergillus fumigatus to the first-line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and propose various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.

Sulfur Metabolism as a Promising Source of New Antifungal Targets.

Fungal infections are a growing threat to human health. Despite their clinical relevance, there is a surprisingly limited availability of clinically approved antifungal agents, which is seriously aggravated by the recent appearance and fast spread of drug resistance. It is therefore clear that there is an urgent need for novel and efficient antifungals. In this context, metabolism is recognized as a promising source for new antifungal targets and, indeed, there are new drugs in development that target metabolic pathways. Fungal sulfur metabolism is particularly interesting, as many of its processes are essential for viability and/or pathogenicity and it shows substantial differences with human metabolism. This short-review will summarize our current knowledge of sulfur-related genes and routes that are important for Aspergillus fumigatus virulence, which consequently could be pursued for drug development.

The Caspofungin Paradoxical Effect is a Tolerant "Eagle Effect" in the Filamentous Fungal Pathogen Aspergillus fumigatus.

Cell responses against antifungals other than resistance have rarely been studied in filamentous fungi, while terms such as tolerance and persistence are well-described for bacteria and increasingly examined in yeast-like organisms. Aspergillus fumigatus is a filamentous fungal pathogen that causes a disease named aspergillosis, for which caspofungin (CAS), a fungistatic drug, is used as a second-line therapy. Some A. fumigatus clinical isolates can survive and grow in CAS concentrations above the minimum effective concentration (MEC), a phenomenon known as "caspofungin paradoxical effect" (CPE). Here, we evaluated the CPE in 67 A. fumigatus clinical isolates by calculating recovery rate (RR) values, where isolates with an RR of ≥0.1 were considered CPE+ while isolates with an RR of <0.1 were classified as CPE-. Conidia produced by three CPE+ clinical isolates, CEA17 (RR = 0.42), Af293 (0.59), and CM7555 (0.38), all showed the ability to grow in high levels of CAS, while all conidia produced by the CPE- isolate IFM61407 (RR = 0.00) showed no evidence of paradoxical growth. Given the importance of the calcium/calcineurin/transcription factor-CrzA pathway in CPE regulation, we also demonstrated that all ΔcrzACEA17 (CPE+) conidia exhibited CPE while 100% of ΔcrzAAf293 (CPE-) did not exhibit CPE. Because all spores derived from an individual strain were phenotypically indistinct with respect to CPE, it is likely that CPE is a genetically encoded adaptive trait that should be considered an antifungal-tolerant phenotype. Because the RR parameter showed that the strength of the CPE was not uniform between strains, we propose that the mechanisms which govern this phenomenon are multifactorial. IMPORTANCE The "Eagle effect," initially described for bacterial species, which reflects the capacity of some strains to growth above the minimum inhibitory concentration (MIC) of specific antimicrobial agents, has been known for more than 70 years. However, its underlying mechanism of action in fungi is not fully understood and its connection with other phenomena such as tolerance or persistence is not clear yet. Here, based on the characterization of the "caspofungin paradoxical effect" in several Aspergillus fumigatus clinical isolates, we demonstrate that all conidia from A. fumigatus CPE+ strains are able to grow in high levels of the drug while all conidia produced by CPE- strains show no evidence of paradoxical growth. This work fills a gap in the understanding of this multifactorial phenomenon by proposing that CPE in A. fumigatus should be considered a tolerant but not persistent phenotype.

Antagonism of the Azoles to Olorofim and Cross-Resistance Are Governed by Linked Transcriptional Networks in Aspergillus fumigatus.

Aspergillosis, in its various manifestations, is a major cause of morbidity and mortality. Very few classes of antifungal drugs have been approved for clinical use to treat these diseases and resistance to the first-line therapeutic class, the triazoles are increasing. A new class of antifungals that target pyrimidine biosynthesis, the orotomides, are currently in development with the first compound in this class, olorofim in late-stage clinical trials. In this study, we identified an antagonistic action of the triazoles on the action of olorofim. We showed that this antagonism was the result of an azole-induced upregulation of the pyrimidine biosynthesis pathway. Intriguingly, we showed that loss of function in the higher order transcription factor, HapB a member of the heterotrimeric HapB/C/E (CBC) complex or the regulator of nitrogen metabolic genes AreA, led to cross-resistance to both the azoles and olorofim, indicating that factors that govern resistance were under common regulatory control. However, the loss of azole-induced antagonism required decoupling of the pyrimidine biosynthetic pathway in a manner independent of the action of a single transcription factor. Our study provided evidence for complex transcriptional crosstalk between the pyrimidine and ergosterol biosynthetic pathways. IMPORTANCE Aspergillosis is a spectrum of diseases and a major cause of morbidity and mortality. To treat these diseases, there are a few classes of antifungal drugs approved for clinical use. Resistance to the first line treatment, the azoles, is increasing. The first antifungal, olorofim, which is in the novel class of orotomides, is currently in development. Here, we showed an antagonistic effect between the azoles and olorofim, which was a result of dysregulation of the pyrimidine pathway, the target of olorofim, and the ergosterol biosynthesis pathway, the target of the azoles.

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.

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.