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.
Aspergillus fumigatus is an opportunistic fungal pathogen that may cause invasive infections in immunocompromised patients. During the last decades the incidence rate of invasive pulmonary aspergillosis (IPA), the most severe infection caused by this mould [1], has dramatically increased which mainly results from the rise in immune-suppressive medical therapies. Furthermore, IPA is accompanied by a high mortality rate which may reach up to 90% depending on the immune status of the host [2], [3], [4]. This pronounced lethality is primarily attributed to the relative inefficiency of current chemotherapies [5], which are based on disrupting the integrity of the fungal cell membrane or cell wall [6]. Therefore, an urgent need to identify targets for the development of novel antifungal substances is evident. Nutritional supply is an essential prerequisite for the onset and manifestation of infection by any pathogen [7]. In opportunistic fungi like A. fumigatus, which does not seem to express host-specific virulence factors [8], [9], nutrient uptake and metabolic versatility have to be considered as non-specific virulence determinants (for a recent review see [10]) that, however, might represent promising antifungal targets [11]. To date, several metabolic routes fundamental for IPA manifestation have been identified: de novo UMP biosynthesis [12], the folate synthesis route [13], siderophore-mediated iron assimilation [14], or the methylcitrate cycle [15] are essential metabolic processes supporting in vivo growth and virulence of A. fumigatus [16]. Nevertheless, detailed knowledge about the metabolic status of the fungus during intrapulmonary growth is still scarce due to the complexity of the pathogen-host system. In a seminal study, preliminary insights into the A. fumigatus-host adaptation transcriptome were gained employing extensive transcriptional profiling under various culture conditions and during an early phase of pulmonary infection, revealing the main stressors encountered by the fungal pathogen when germinating in a susceptible, leukopenic mammalian host [17]. Among these, starvation for nitrogen became evident. Yet, while numerous studies have focused on A. fumigatus nitrogen metabolism [16], [18], [19], [20], [21], [22], neither the exact source (s) of this macroelement nor specific metabolic routes of nitrogen assimilation during pulmonary infection have been identified so far. Sulphur (S) is another essential nutrient that the fungus needs to acquire from the surrounding tissue during intrapulmonary growth, as it is a constituent of the proteinogenic amino acids cysteine and methionine as well as of essential organic molecules like coenzyme-A, glutathione and, particularly, iron-sulphur (Fe-S) clusters. Only a few studies have addressed its relevance for fungal virulence so far, focusing on synthesis and utilization of the sulphur-containing molecule glutathione in Candida albicans or C. glabrata [23], [24]. The importance of sulphur metabolism for aspergillosis has not been addressed to date. Aspergillus species can utilize a variety of sulphur-containing molecules, such as inorganic S-sources, e. g. sulphate, or sulphate esters of organic compounds [25]. The assimilation of sulphate is performed via a well-defined pathway that comprises its uptake by specialised permeases, activation by ATP-driven phosphorylation in two steps, and reduction to sulphite and further to sulphide [26]. The latter is condensed with O-acetyl serine to yield cysteine, from which methionine and also S-adenosylmethionine can be formed. Sulphur homeostasis is tightly regulated in filamentous fungi by catabolite repression [27]. In the model filamentous fungus Neurospora crassa for instance, CYS-3, a positive-acting transcriptional factor of the bZIP family, was identified that activates expression of a set of genes whose products are required to acquire and utilize secondary S-sources under sulphur-starved conditions [28], [29], [30]. Furthermore, the role of so-called sulphur controller (scon) genes has been addressed that influence methionine repression of sulphur-containing amino acid biosynthesis [31]. Regulation of sulphur metabolism has also been extensively studied in the bakers' yeast Saccharomyces cerevisiae (for detailed information consult reviews [32] or [26]). While in N. crassa CYS-3 is sufficient to bind DNA and to activate transcription, in S. cerevisiae a heteromeric complex of three proteins (Met4-Met28-Cbf1) is required for proper sulphur-dependent regulation [33], [34], [35]. The CYS-3 orthologue of A. nidulans, MetR, is required to activate transcription of several genes related to sulphur acquisition and to allow growth on a variety of sulphur sources other than methionine [36]. Successful complementation of an A. nidulans metRΔ strain with its MET1 orthologue from the dimorphic human-pathogenic fungus Paracoccidioides brasiliensis demonstrated functional conservation of this transcriptional regulator [37]. Adaptation to environmental stressors is usually based on reprogramming of the cellular expression pattern triggered by specific transcription factors. Consequently, the relevance of several cellular processes for A. fumigatus virulence has been investigated by targeting the corresponding regulators (for review see ref. [38]). Deletion of genes coding for zinc or iron responsive factors demonstrated the importance of both elements for virulence [39], [40]. Also, a positive role of amino acid homeostasis in A. fumigatus virulence was established by deletion of the respective cross-pathway control regulator [41]. A substantial benefit from targeting wide-domain regulators of a given cellular aspect lies in its comprehensive outcome, in contrast to particular gene deletions affecting activities that may be encoded redundantly in the fungal genome. Based on the hypothesis that flexible regulation of sulphur homeostasis supports growth in a susceptible host and therefore might affect virulence of A. fumigatus, we became interested in the cellular function of the MetR orthologue in this opportunistic pathogen. Our results demonstrate that this transcription factor is a key regulator of sulphur assimilation and that it is crucial for A. fumigatus pathogenicity. Moreover, we describe an unprecedented regulatory crosstalk of S-metabolism and iron homeostasis.
The A. fumigatus MetR transcription factor was identified by BLAST search [42], [43], [44] on the NCBI server (http: //www. ncbi. nlm. nih. gov/) using the A. nidulans MetR protein sequence (PubMed acc. no. AAD38380) as a query, revealing 64% identity and 75% similarity between both proteins (Fig. 1A). Notably, the leucine zipper (bZIP) domains of the factors are virtually identical with 97% identity and 100% similarity, which suggests that both could recognize a similar DNA target sequence. In the CADRE database [45] the A. fumigatus metR gene (AFUA_4G06530) had been automatically annotated to contain 1517 base pairs (bp), resulting in a predicted coding region of 918 nucleotides (nt) based on the presence of an unusually long intron 599 nt in size. The observable size difference in a 1% agarose gel between a metR cDNA (obtained by reverse transcription from mRNA) and gDNA (amplified from genomic DNA) matched with the presence of this intron (not shown), and sequencing of a complete cDNA insert confirmed the overall architecture of the metR gene as annotated. The deduced protein sequence comprises 305 amino acids with a predicted molecular weight of 33 kDa. The MetR transcription factor displays a high degree of conservation among ascomycetous fungi (Fig. 1B), being present in all genera analysed. With respect to the A. fumigatus MetR sequence, identities/similarities range from 25/33% to the N. crassa orthologue and up to 95/100% to the Neosartorya fischeri one. Also, the unusually long intron appears to be conserved among ascomycota, suggesting a common origin of the gene. With the aim to probe localisation as well as any cellular function of the metR gene product, an A. fumigatus strain that would express a functional GFP-tagged version from the endogenous metR promoter was constructed. This strain AfS171 was shifted from sulphate-containing medium for 90 minutes to medium lacking any source of sulphur (Fig. 1C). Under S-rich conditions a faint fluorescent signal was observed that was uniformly distributed in the hyphal cytoplasm. Upon S-depletion, however, translocation of the MetR-GFP protein to the nuclei became evident, as deduced from co-localisation of the fluorescent signals with the nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI). Accordingly, the presumed DNA binding domain of MetR and its cytoplasmic-nuclear shuttling under sulphur-limiting conditions imply that this gene product acts as a transcriptional regulator of A. fumigatus sulphur metabolism. To gain insights into the cellular function of the metR gene product, a full deletion strain of A. fumigatus was constructed by homologous gene replacement employing a self-excising recyclable cassette that contains a hygromycin B resistance gene as selectable marker [46], [47]. Southern analysis of the resulting strains AfS166 [metR: : six-β-rec/hygroR-six] and AfS167 [metR: : six] confirmed the homologous replacement and the excising event, respectively (Fig. S1A). A preliminary phenotypic analysis revealed that a metR deletant is unable to grow in the presence of sulphate as sole source of sulphur (Fig. 2A). This allowed us to reintroduce the metR gene at its original locus without using any selection marker but the presence of sulphate (SO42−) as the only source of sulphur. In order to differentiate between the desired reconstituted strain and its wild-type progenitor, a silent mutation was introduced in the gene' s coding sequence to create an additional BstEII restriction site. Southern analysis confirmed the correct integration event for a representative isolate (Fig. S1B). To address the role of the MetR factor for the ability of A. fumigatus to utilize different sulphur sources, the resulting strains were subjected to phenotypic inspection on various S-containing media (Fig. 2A). With respect to inorganic S-sources, the metR: : six (syn. metRΔ) mutant strain AfS167 was unable to grow in the presence of any of the tested substrates. Among the organic sources, the metRΔ mutant grew on methionine as well as homocysteine, which contrasts with a corresponding A. nidulans metRΔ mutant that was described to grow only poorly on the latter compound [32]. It was noticed, however, that homocysteine did not suffice as source of sulphur at alkaline pH while methionine was perfectly assimilated (not shown). Furthermore, AfS167 was able to utilize cysteine or glutathione as combined nitrogen and sulphur sources when the culture media were additionally depleted for nitrogen. Importantly, the metRΔ strain barely grew on porcine lung agar (PLA), suggesting that this mutant could suffer from a growth defect within the pulmonary tissue of a susceptible host and, therefore, might have reduced virulence capacities. When methionine was added to the PLA medium growth of the mutant was restored, which indicates that this tissue contains insufficient free levels of this amino acid to support growth of the deletant. In order to test for any cross-talk of utilisation pathways for other macro-elements, growth of the metRΔ mutant was monitored on various N-, C-, or P-sources in the presence of methionine and sulphate, respectively (Fig. S2). With one exception, no obvious phenotype emerged in dependency of the various supplements, i. e. the mutant was able to grow only when methionine was present as source of sulphur. However when galactose served as C-source, growth of the deletant appeared repressed even in the presence of methionine, which indicates that this particular sugar interferes with methionine utilisation in A. fumigatus. To better understand the growth defect of AfS167 on inorganic S-sources, germination of the deletant was investigated (Fig. 2B). The strain was unable to swell or germinate in the presence of SO42− up to eleven hours after inoculation; on the contrary, its germination on methionine was only slightly delayed in comparison with the wild-type strain and reached approximately the same rate after prolonged incubation. Therefore, it appears that sulphur assimilation is an indispensable prerequisite for proper germ tube emergence and its absence results in a pronounced germination and growth defect as displayed by the metR: : six mutant strain. To further analyse the S-assimilation capacity of the deletion strain we made use of Biolog Phenotype MicroArrays (Fig. 3). This technology allows the evaluation of a variety of microbial phenotypes in a parallel fashion. From a variety of conditions and stressors available, the PM4 MicroPlate plate containing 35 different sulphur sources was selected. Wild-type and mutant strains were incubated in parallel in the wells of this microtiter plate and their growth ability in the presence of different S-sources was monitored via the optical density (O. D.) at 630 nm. The recommended Biolog Redox Dye to monitor growth by measuring the respiration process was also tested; however, as previously described for filamentous fungi [48], the results obtained using this dye turned out to be inconsistent (data not shown). Therefore, one well lacking any sulphur source was used to normalize O. D. values to define a growth threshold of 0. 2, which was further confirmed through microscopic inspection. Since the use of phenotypic microarray plates is a technology that has not been extensively used for filamentous fungi yet, some of the compounds present in the PM4 MicroPlate were re-tested in regular phenotypic assays on solid culture media to support validity of the results (not shown). Growth phenotypes deduced from this Biolog plate confirmed that the metRΔ strain is unable to utilise any oxidized inorganic sulphur source. Indeed, it only grew on methionine or derivatives thereof (i. e. N-acetylmethionine or methionine sulphoxide). Notably, the mutant did not grow in the presence of methionine sulphone, although the wild-type strain grew perfectly well, but it grew better than its progenitor on N-acetylmethionine. The central conclusions reached from these phenotypic analyses are that the metRΔ mutant is unable to grow on inorganic sulphur sources and that utilization of methionine and its derivatives is independent from the presence of the transcriptional activator MetR. Because the AfS167 strain is unable to grow in the presence of S2− (Fig. 2A) and as it is known that several fungi, including Aspergillus species, produce volatile sulphur compounds (VSCs) like hydrogen sulphide (H2S), dimethylsulphide (H3C-S-CH3), or methanethiol (CH3-SH) as a result of methionine catabolism [49], [50], [51], we became interested in studying whether A. fumigatus would be able to utilize such volatile compounds as S-source and if generation of such VSCs is MetR-dependent. The wild-type and metRΔ strains were cultured in small petri dishes with minimal medium containing methionine as sulphur source. These plates were placed inside larger petri dishes with medium lacking any S-source. Neither the wild-type isolate nor the mutant was able to grow in the absence of any sulphur source (not shown). However, when either strain was grown on the methionine-containing petri dish, growth on the outside sulphur-depleted medium was observed only for the wild-type strain (Fig. 4). Accordingly, A. fumigatus is able to take up VSCs produced from methionine catabolism and to use them as S-source. Production of VSCs appears to be independent from the presence of the MetR regulator but their utilization as S-source requires the presence of this regulatory factor. In the light of these results, it is valid to conclude that the MetR factor represents a master regulator of sulphur assimilation in A. fumigatus acting predominantly on utilization of inorganic S-sources, while consumption of methionine does not depend on this transcriptional regulator. The aforementioned phenotypic results suggest that the MetR factor might activate the transcription of genes required for the uptake and utilization of different sulphur sources. To investigate this, the short-term transcriptional responses of a wild-type strain and its metRΔ derivative to variations in the available sulphur source were monitored: Mycelia grown overnight in Aspergillus minimal medium (AMM) with methionine were shifted to media containing diverse S-sources, incubated for one additional hour, and steady-state levels of several transcripts of genes related to sulphur uptake and utilization were monitored by Northern blot hybridisation. Initially, transcript levels of the metR gene itself were checked to observe that its transcription is apparently not regulated by the nature of the sulphur source (Fig. 5A), resembling the situation in A. nidulans [32] but contrasting findings in N. crassa [24], [25]. Interestingly, a second hybridising signal was detected for the metR transcript under S-starvation conditions, indicating alternative processing of the encoding transcript. In order to further understand why the metRΔ mutant is unable to grow on oxidized inorganic sulphur sources, expression of all genes encoding enzymes of the sulphate assimilation pathway, which are sulphate permease (sB), ATP-sulphurylase (sC), APS-kinase (sD), PAPS-reductase (sA) and sulphite reductase, as well as expression of one arylsulphatase-encoding gene (required for utilization of sulphur esters, i. e. nitrophenyl sulphate) was checked (Fig. 5A). Upregulation of the sulphate permease-, ATP-sulphurylase- and APS-kinase-encoding genes under sulphur-starving conditions depended on the presence of MetR factor. For the sB gene, a second, longer transcript became evident under sulphur starvation. Transcription levels of the genes coding for PAPS reductase and sulphite reductase were decreased in the absence of the MetR factor on all S-sources in comparison to the wild-type. Furthermore, expression of the arylsulphatase-encoding gene was completely shut down in the mutant. This transcriptional pattern agrees with and partially explains the incapacity of the metRΔ deletant to grow on inorganic sulphur sources. In contrast to inorganic sulphur sources, the AfS167 deletion mutant grows on methionine independently of other conditions such as varying pH (not shown) or the availability of nitrogen. To address the reason for this phenotype, transcription of several methionine-related genes was investigated (Fig. 5B). A BLAST search against the A. fumigatus genome sequence identified three putative transporters involved in methionine uptake, which we named mup genes. Transcript levels of mupA and mupC were constitutive with respect to the sulphur source and seemed to be mostly independent of MetR, although mupA expression appeared somewhat elevated in the wild-type. Transcription of the related mupB gene could not be detected under any condition tested (not shown). Transcription of the putative methionine synthase-encoding gene metH was also not regulated by MetR. Interestingly, its expression was reduced in the presence of methionine and increased under sulphur-starving conditions, suggesting that the intracellular pool of methionine is constantly maintained and emphasizing the importance of this particular amino acid. Importantly, the methionine aminotransferase-encoding gene metAT, whose product is probably responsible for methionine degradation [52], [53] and consequently for its utilization as sulphur source, was highly expressed in the presence of methionine in a MetR-independent manner. Therefore, the expression pattern of these genes perfectly agrees with the ability of the deletion strain to utilize methionine as S-source. Interestingly in the metRΔ mutant, metAT was highly expressed under sulphur-depleted conditions. We hypothesized that this is due to the strong and rapid sulphur starvation affecting the mutant under such sulphur-restricted conditions. To verify this assumption, the wild-type and mutant strains were incubated up to eight hours on sulphate-containing and sulphur-depleted media (Fig. 5B), which results in substantial sulphur-limiting conditions for the deletant but not for the wild-type. Accordingly, metAT expression was upregulated in the metRΔ mutant but not in the wild-type, demonstrating that after prolonged incubation in the presence of sulphate the mutant strain becomes depleted for sulphur. Surprisingly, the wild-type did not upregulate expression of metAT even after eight hours of incubation under sulphur-depleted conditions, implying that the resulting sulphur starvation in the wild-type is not that severe, probably due to MetR-dependent recycling processes and mobilization of reserves. In conclusion, the metAT expression profile suggests that other mechanisms apart from MetR-mediated regulation must exist to orchestrate gene expression depending on the availability and source of sulphur. With respect to cysteine assimilation no clear candidates for its degradation and utilization as sulphur source have been identified so far and, consequently, the incapacity of the deletion strain AfS167 to grow on cysteine could not be addressed properly. Nevertheless, expression of the putative cysteine transporter-encoding gene cynA, an orthologue to a C. glabrata-specific cysteine transporter [54], was not upregulated in the metRΔ strain under sulphur starving conditions (Fig. 5C), which would partially explain the observed phenotype. Furthermore, transcription of the cysteine synthase-encoding gene cysB was slightly increased in the absence of cysteine in a MetR-dependent manner, which might translate into a slightly reduced level of cysteine in the deletion strain. To demonstrate a direct effect of MetR on the transcription of sulphur assimilation genes, we performed chromatin immuneprecipitation (ChIP) analyses making use of strain AfS171 expressing a functional GFP-tagged version of this transcription factor. Fixed chromatin samples isolated from fungal cultures that had been starved for sulphur were sheared and precipitated with a nano-trap (see Supporting Information for details). Interrogating the output fractions by semi-quantitative PCR revealed a pronounced and reproducible enrichment of fragments spanning the promoters of several candidate genes, such as the ones encoding the APS-kinase, the arylsulphatase, the ATP-sulphurylase, as well as the sulphate permease (Fig. 5D). Following the observation that the expression of genes whose products are required for sulphur assimilation is regulated by MetR, we became interested in understanding to what extent any transcriptional remodeling that takes place under sulphur-limiting conditions is MetR-dependent. For this purpose, overnight-grown mycelia of the wild-type and metRΔ strains were shifted from cultures containing sufficient levels (5 mM) of methionine serving as sole S-source to media containing low methionine levels (0. 2 mM) over a time frame of eight hours before RNA was harvested. Previous culturing experiments had shown that methionine depletion became manifest within this time frame, so this experimental set-up allows assessment of any MetR contribution to the transcriptional response upon mild S-depletion. Nucleic acid samples were prepared from two biological replicates each to perform digital transcriptome analyses by the RNA-seq approach (see Materials and Methods for details). Comparison of both transcriptomes under this specific condition revealed that 288 genes were downregulated and 349 were upregulated in the metRΔ strain with respect to its wild-type progenitor (>1. 5-fold change, p-value<0. 05) (Table 1 and Table S1). Categorisation via the FungiFun suite [55] revealed that the main cellular functions affected by the absence of MetR are membrane transport, metabolism, carbohydrate metabolism, and oxidation/reduction (Fig. 6). Therefore, MetR action is required for the correct remodeling of these processes to counteract conditions of sulphur depletion. To further understand this adaptation, we performed a deeper functional categorisation. Various genes assigned to cation homeostasis were less abundant in the metRΔ mutant (Table 2), suggesting a strong dysfunction in the regulation of the metabolism of these ions. In addition, several genes whose products participate in sugar, glucoside, polyol and carboxylate metabolism were downregulated what highlights the greatly different metabolic status of the mutant under sulphur starvation. Furthermore, genes related to cellular export and secretion were also identified, hinting a distinct interaction with the environment. Surprisingly, several genes related to mRNA synthesis and were also found to be downregulated, which indicates a interplay of MetR regulation with other transcription factors and cell cycle regulation. Among the transcripts that are more abundant in the mutant (Table 3), several genes whose products participate in amino acid transport were of special interest as this implies a link between nitrogen and sulphur assimilation. In addition, various genes related to DNA conformation and repair were found to be upregulated, which might reflect the severe stress situation for the mutant under sulphur starving conditions. Intriguingly, several genes related to cation transport, and especially siderophore transport and reductive iron assimilation, were found to be upregulated, suggesting a connection of MetR-mediated regulation to iron homeostasis. Indeed, among the 20 genes that showed a higher expression in the mutant compared to the wild-type (Table 1), 11 genes that have previously been found to be upregulated during iron starvation dependent on the iron regulator SreA were identified, including five genes of proven function in siderophore biosynthesis [56], [57]. Further inspection of the entire list of upregulated genes revealed 29 of the known 49 SreA target genes with 13 of proven function in siderophore metabolism, reductive iron assimilation and iron regulation (not shown). In conclusion, the MetR-dependent adaptation to sulphur starvation conditions is a complex process that involves broad transcriptional remodeling to achieve altered expression of genes belonging to various functional categories. In order to address the role of fungal sulphur utilization for growth and therefore virulence in a susceptible host, the involvement of the sulphur-related transcriptional regulator MetR in A. fumigatus virulence was assessed in different animal models. Initially, the alternative host model of the wax moth larvae Galleria mellonella was assayed (Fig. S3), where the metRΔ mutant displayed a significantly reduced virulence (p-value of <0. 001) similar to the reduction observed for the control strain, an avirulent pabaAΔ mutant. This decrease in virulence was specifically attributed to the absence of metR, since the reconstituted strain recovered full virulence. Interestingly, when injected in a solution containing 5 mM methionine, the metRΔ strain was able to kill larvae as the wild-type, suggesting that the decrease in virulence is due to the absence of a proper source of sulphur. The results obtained in the wax moth model encouraged us to perform infections in established mouse models of aspergillosis. When challenging immunosuppressed, leukopenic mice intranasally with conidia of the metRΔ mutant to induce invasive pulmonary aspergillosis, a highly significant (p<0. 001) reduction in the virulence capacity of this strain was observed (Fig. 7A) with more than 80% of the cohort surviving the infection. This virulence attenuation was once again specifically ascribed to the absence of metR, since the reconstituted strain regained full virulence. Histological inspection of lung sections from infected animals revealed that infectious propagules of the metRΔ strain had been cleared in the course of infection by the residual immune system, in contrast to invasive tissue penetration of the wild-type progenitor strain. Accordingly, all lungs inspected from the metRΔ-infected cohort appeared as normal, while invasive growth of hyphal elements became evident for those infected with the wild-type strain. Fungal burdens assessed from lungs of infected mice (n = 5 animals per group) indicated a 50-fold reduction in colony forming units per gram tissue (500±141 vs. 23 849±3 770) for the deletion strain in comparison to its wild-type progenitor (Fig. 7B). To further corroborate the differences in virulence, competitive infection experiments were performed in order to obtain a competitive index (CI) [13]. In this assay, a cohort of four animals was infected with an input ratio of 1∶1 for wild-type and the deletion mutant and, four days later, output ratios were determined by assessing the number of colony forming units on permissive and selective media from homogenised pulmonary tissue. A mean CI value smaller than 0. 1 was calculated for the metRΔ mutant (Fig. 7C), meaning that this strain is virtually avirulent. To finally analyse the dissemination capacity of the metRΔ mutant in the bloodstream of leukopenic mice and the relevance of sulphur utilization for this process, a systemic infection model was applied (Fig. 7D). Animals infected intravenously with the mutant strain showed significantly delayed mortality (p<0. 001). In the light of these results we conclude that regulation of sulphur assimilation is essential for manifestation of pulmonary aspergillosis as well as relevant for haematogenous dissemination after angioinvasion of A. fumigatus. It is well established that defects in mitochondrial Fe-S cluster biogenesis or transport induce transcription of the iron regulon [58], [59] and that Fe-S cluster-containing proteins participate directly in sensing iron availability in S. cerevisiae [60], [61]. Accordingly, we expected a regulatory cross-talk between sulphur assimilation and iron homeostasis in A. fumigatus, which was further indicated by the transcriptional profiling data (see above), however, which had not been tested in eukaryotes so far. We took advantage of our metRΔ mutant strain, which can be rapidly depleted for sulphur, to test this hypothesis. Overnight grown strains were shifted from culture media containing methionine to media depleted for this amino acid but containing sulphate. These media pose sulphur starving-conditions for the deletion strain but not for the wild-type or the reconstituted strain. After eight hours of incubation, although there was sufficient iron in the medium, the mutant strain increased transcription of several genes encoding proteins that participate in iron acquisition that are known to be upregulated under iron starvation, i. e. genes involved in siderophore biosynthesis (sidA), siderophore transport (mirB), mitochondrial ornithine export (amcA) and iron regulation (hapX) [57]. In contrast, the mutant decreased transcription of genes whose products participate in iron-consuming processes that are known to be downregulated under iron depleted conditions [40], i. e. genes encoding aconitase (acoA), cytochrome c (cycA) or components of the mitochondrial iron-sulfur-cluster biosynthetic machinery (isa1) (Fig. 8A). To analyse whether the cells were indeed depleted for iron, levels of iron chelated by ferricrocin (FC), the intracellular siderophore used for iron storage and transport [62], [63], were measured (Fig. 8B). Despite an expression pattern resembling that of iron starvation, the AfS167 mutant showed a nearly fivefold increased FC content. Combining ferricrocin analysis with total intracellular iron level measurements further underscored this imbalance in iron homeostasis (Table 4): MetR deficiency raises the cellular iron content 1. 6-fold in the presence of methionine, which increases to 2. 8-fold in its absence. The metRΔ mutant furthermore displays a 5-fold increased FC-chelated iron content under +Met conditions that is further enhanced to 7. 5-fold when this S-sources is withdrawn. In conclusion, the cells indeed contain sufficient amounts of iron but display a defect in iron sensing and/or regulation. This dysregulation, causing an enhanced expression of iron uptake-related genes under sulphur starving conditions, translates into a phenotype of hypersensitivity to iron (Fig. 8C). At a low concentration of iron and methionine the metRΔ mutant was able to grow, although poorly due to the shortage of sulphur. This phenotype was recovered with higher availability of methionine. However, at higher iron concentrations, the wild-type strain could grow while the AfS167 mutant did not, unless a high amount of methionine was present in the medium. This might be the consequence of both the inability to shut down expression of iron uptake-related genes and of the lower cccA gene expression, encoding a recently described vacuolar transporter that has a prominent role in iron detoxification [64], in the metRΔ mutant especially under sulphur starvation (Fig. 8A), which results in iron accumulation (see Table 4: 2. 4-fold increase for the metRΔ deletant under –Met conditions) to presumably toxic levels in the cytosol. Dysregulation of iron homeostasis was also tested in the wild-type strain by shifting its mycelium to a medium completely depleted for sulphur. However, expression of the iron regulon was not observed even after 24 hours of incubation (not shown), most likely because of the fact that the wild-type does not face such severe sulphur starving conditions as its metRΔ derivative apparently does.
Fulfillment of nutritional and metabolic requirements is essential for all pathogenic microorganisms to be able to grow inside the host and, thus, to cause infection and disease [7]. For opportunistic fungal pathogens this is highly relevant, since these commonly lack specific virulence factors that would provoke host damage [9]. In recent years more and more evidence has been provided that fungal metabolism is a critical component of fungal virulence [65]. Accordingly, it has been proposed that based on this knowledge novel antifungal targets might be identified [10]. Sulphur metabolism is directly related to virulence of several pathogenic microorganisms, such as Mycobacterium tuberculosis, Salmonella enterica, or protozoan parasites [66], [67], [68], [69], [70], [71]. Among fungi, sulphur metabolism has been extensively studied in the bakers' yeast S. cerevisiae [30], [32] and in the non-pathogenic filamentous fungi N. crassa [24], [28], [29] and A. nidulans [36]. However, our knowledge on the role of sulphur metabolism for fungal virulence has remained scarce. Only two studies have specifically addressed the importance of the sulphur-containing molecule glutathione in C. albicans and C. glabrata to demonstrate that glutathione biosynthesis, but not its uptake or degradation, is essential for virulence [23]. Accordingly, glutathione appears not to be the sulphur source these Candida species exploit in vivo, and its relevance for pathogenesis is probably due to its impact on iron metabolism [72]. For the human pathogen Paracoccidioides brasiliensis it was demonstrated that growth of the yeast form, which is the pathogenic state of this dimorphic fungus, strictly relies on inorganic sulphur sources and that the mycelial-to-yeast switch requires an organic source of sulphur [37], [73]. Here, we report first evidence that proper regulation of sulphur metabolism is crucial for A. fumigatus virulence. This result can possibly be extrapolated to other fungal pathogens and, therefore, might constitute a novel field for the identification of new targets in fighting fungal infections. The information gathered in this work suggests that the MetR regulon directly affects genes whose products are related to assimilation processes of sulphur (especially inorganic sulphur), rather than metabolic processes (Fig. 9). Presence of the MetR transcription factor is essential for growth on several sulphur sources, specifically on those containing inorganic sulphur sources. Accordingly, MetR is required for activation of transcription of genes encoding enzymes of the sulphate assimilation pathway and an arylsulphatase activity, which demonstrates a direct role of MetR in inorganic sulphur acquisition. Remarkably, the metRΔ mutant was able to use cysteine and the Glu-Cys-Gly tripeptide glutathione as a source of sulphur only under nitrogen-starving conditions, implicating a link between S- and N-acquisition. One possible explanation is that under nitrogen-limiting conditions increased expression of amino acid permeases and oligopeptide transporters facilitates uptake of these particular sulphur-containing compounds, which then can be exploited as S-source. Since no specific enzymes for cysteine catabolism have been identified so far, this scenario could not be investigated further. The fact that the expression of the methionine aminotransferase-encoding gene metAT is elevated in the presence of cysteine suggests that this amino acid might be transformed into methionine rather than being catabolized directly. Accordingly, catabolism of cysteine as sulphur source appears to be MetR-independent and, therefore, its uptake might represent a bottle neck that prevents the mutant to utilize cysteine. This notion is further supported by the fact that in the RNA-seq data set expression of the oligopeptide transporter OptG (AFUA_6G03140), the orthologue to the C. albicans glutathione transporter OPT7 [23], was observed to be expressed higher in the wild-type than in the metRΔ mutant. Thus, cynA and optG are candidate genes to support A. fumigatus growth in the presence of cysteine and glutathione, but further studies are needed to elucidate whether they encode specific A. fumigatus transporters and whether cysteine acid is catabolized directly. The ability of A. fumigatus to produce volatile sulphur compounds derived from methionine catabolism has been demonstrated previously [51]. Here we demonstrate for the first time that A. fumigatus is furthermore able to utilize such VSCs as S-source. Importantly, we could show that utilization but not production of VSCs is MetR-dependent. This agrees with the inability of the metRΔ mutant to grow on S2− and also with the fact that all studied genes that participate in methionine metabolism are MetR-independent in their expression. Nevertheless, further studies are necessary to unravel the methionine catabolism pathway in order to understand the capacity of a metRΔ mutant to utilize it as S-source, the VSCs production process, and also to identify any specific VSCs that can be utilized. As an important part of our current study we could show that MetR is important for virulence of A. fumigatus in G. mellonella larvae as well as in leukopenic mice. In the wax moth larvae, metRΔ regained virulence when supplemented with methionine, suggesting that the main reason for the decrease in virulence is the absence of a suitable source of sulphur in the larval hemocoel. In the same way one might speculate that the decrease in virulence observed in the mouse model is also due to insufficient levels of methionine in the murine lung or blood and, thus, that this amino acid is not the primary source of sulphur exploited by A. fumigatus within the pulmonary tissue or in the bloodstream. In line with this are data from Purnell (1973) on a methionine-requiring mutant of A. nidulans that displayed unaltered virulence in systemic infections of mice [74]. Inorganic compounds that cannot be assimilated by the metRΔ mutant may accordingly serve as initial S-source during infection. Taking into account that the mammalian lung probably constitutes a nitrogen-limiting environment [17], cysteine and glutathione also likely do not serve as sources of sulphur during pulmonary infection, since the mutant is able to utilize these compounds in vitro under nitrogen-starving conditions. However, since several other sulphur-related processes are deregulated in the metRΔ mutant, we cannot conclude that the mere absence of a suitable sulphur source impairs the growth of the metRΔ mutant within the murine lung. Defects in iron regulation or sensing that are characteristic for the metRΔ mutant may also account for its attenuated virulence, since iron relates to fundamental cellular processes such as respiration or oxidative stress resistance [40], [75]. In the bakers' yeast the Aft1p and Aft2p transcription factors mediate upregulation of the so-called iron regulon under iron limiting conditions [76], [77], [78]. Iron sensing by Aft1p and Aft2p requires proper mitochondrial Fe-S cluster biosynthesis as well as a functional export to the cytoplasm and, consequently, disturbance of these processes provokes upregulation of the iron regulon [58], [59], [79]. Impairment of Fe-S cluster biogenesis can be achieved by disruption of the cysteine desulphurase-encoding gene nfs1 that is required for sulphide supply in Fe-S cluster biosynthesis [80], [81]. In addition, it was shown that glutathione participates in Fe-S cluster translocation to the cytoplasm and thus its depletion activates the iron regulon [79], [82]. All these relations between sulphur-containing molecules and iron-dependent transcriptional regulation strongly suggest a connection between sulphur metabolism and iron homeostasis. Other fungi, such as Aspergillus species or Schizosaccharomyces pombe, do not express Aft1/2 orthologues. Here, iron regulation is mediated by the interplay of the unrelated HapX/Php4 and SreA/Fep1 proteins [40], [83], [84]. Yet, the strategy for iron sensing is likely to be conserved in the fungal kingdom, which might link iron homeostasis to sulphur metabolism in general. Here, by virtue of a deletion mutant, we clearly demonstrate this relationship: starving the A. fumigatus metRΔ mutant strain for sulphur results in increased expression of the iron regulon. We hypothesize that the pronounced sulphur starvation of the metRΔ mutant impairs Fe-S cluster biogenesis and/or glutathione biosynthesis, which, in turn, activates the iron regulon. The corresponding wild-type isolate would not act on iron homeostasis under sulphur-depleting conditions as it apparently does not face such a severe starvation, most likely due to the utilization of reserve pools and salvage pathways. We cannot rule out the possibility that MetR directly regulates transcription of an unidentified gene whose product is required for iron sensing or proper iron regulation under sulphur-starving conditions. But given the fact that MetR deficiency strikingly phenocopies a deficiency for the negative iron regulator SreA (increased cellular iron as well as ferricrocin contents accompanied by transcriptional derepression of genes involved in iron acquisition such as siderophore biosynthesis and uptake as well as reductive iron assimilation), such a direct action on the sreA and hapX genes encoding the main players of iron homeostasis in A. fumigatus is unlikely. Along that line, we could not detect binding of the MetR-GFP protein to promoter regions of either gene by chromatin immune-precipitation. The disclosed link between sulphur metabolism and iron homeostasis represents an appealing crosstalk between two fundamental cellular regulatory circuits that calls for further investigation. In summary, we show for the first time that regulation of sulphur metabolism is important for the ability of A. fumigatus to cause disease. Given the conserved nature of sulphur assimilation in the fungal kingdom, its relevance in virulence is likely to be a general feature among pathogenic fungi. Considering that many of these routes are absent in mammals, some of these processes might represent suitable novel targets for antifungal drug development.
Mice were cared for in accordance with the principles outlined by the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (European Treaty Series, no. 123; http: //conventions. coe. int/ Treaty/en/Treaties/Html/123. htm). All infection experiments were carried out in compliance with the German animal protection law in a protocol approved by the Government of Lower Franconia (file number: 55. 2-2531. 01-90/09). The Escherichia coli strain DH5α [85] was used for cloning procedures. Plasmid-carrying E. coli strains were routinely grown at 37°C in LB liquid medium (1% peptone, 0. 5% yeast extract, 0. 5% NaCl) under selective conditions (100 µg•ml−1 ampicillin or 50 µg•ml−1 kanamycin); for growth on plates, 1. 5% agar was added to solidify the medium. All plasmid constructs used in the course of this study are listed in Table S2 and were generated using the Seamless Cloning (Invitrogen) technology as described in the Supplementary Material (Text S1). The wild-type A. fumigatus strain ATCC 46645 served as common reference [86], derivatives of this isolate generated in the course of this study are described in the Supplementary Material (Text S2). A. fumigatus strains were basically cultured in nitrate-based minimal medium [87] containing 1% glucose, 70 mM NaNO3,7 mM KCl, 11 mM KH2PO4 (pH 5. 5), 0. 25 mM MgSO4, trace elements solution, and 2% agar (Serva) for solid media at 37°C. In case of selection for the presence of the hygromycin B resistance marker, 50 µg•ml−1 of this antibiotic (InvivoGen) were applied. In sulphur-free medium, MgCl2 was substituted for MgSO4 and a modified mixture of trace elements lacking any sulphate salt was used. For preparation of porcine lung agar (PLA) culture medium, 5 g of fresh tissue was snap-frozen in liquid nitrogen and pulverized using a pre-cooled mortar. The resulting powder was put into a 50 ml reaction tube, filled up with an equal amount of sterile saline and briefly incubated in a 50°C warm water bath, followed by addition of an equal amount of 50°C warm, liquid water agar (1. 5% agarose in water). To suppress bacterial growth, PLA media was supplemented with 50 µg•ml−1 tetracycline. The suspension was finally vortexed for 10 s and poured onto solidified water agar. For all growth assays on solid media, the culture medium was inoculated with 10 µl of a freshly prepared A. fumigatus spore suspension (105 conidia•ml−1 in water supplemented with 0. 9% NaCl and 0. 02% Tween 80) and incubated at 37°C for three days. Phenotypic microarray plates (Biolog PM 4) were inoculated as follows: a suspension containing 1. 5×106 spores•ml−1 in sulphur-free minimal medium was prepared and 100 µl aliquots of this were added to each well. Fungal growth was measured at 48 hours by optical density (O. D.) at 630 nm in a Multiskan Ascent microplate photometer (Thermo Electron). For the measurement of germination percentages, 107 conidia were inoculated in 200 ml sulphur-free minimal media supplemented with 2 mM sulphate or 5 mM methionine and incubated at 37°C and 150 r. p. m for 11 hours. Each hour a 1 ml aliquot was taken, sonicated, and the germination percentage calculated as the ratio of germinated conidia with respect to the total number of spores. A. fumigatus liquid media shifts were performed according to Narendja et al. [88] with adjusted media to modify the sources of sulphur: 200 ml minimal medium lacking sulphur and supplemented with 5 mM methionine were inoculated with 108 freshly harvested, 5-day-old A. fumigatus ATCC 46645, metRΔ, or metR+ conidia and propagated at 37°C and 150 r. p. m. for 16 to 22 hours. Mycelia from such pre-cultures were then harvested, washed extensively with water, and split into similar aliquots on a sterile surface. These were then added to 100 ml of minimal medium base without sulphur source or supplemented with 2 mM SO42−, 5 mM methionine, or 5 mM cysteine, respectively, and incubated at 37°C and 150 r. p. m for one to eight hours. Standard protocols of recombinant DNA technology were carried out [89]. Phusion high-fidelity DNA polymerase (Fermentas) was generally used in polymerase chain reactions and essential cloning steps were verified by sequencing. Fungal genomic DNA was prepared following the protocol of Kolar et al. [90] and Southern analyses were carried out as described [91], [92]. Probes for non-radioactive hybridizations were generated and detected using the Gene Images AlkPhos Direct Labelling and Detection System from GE Healthcare. Samples of total RNA were isolated with the TRIzol reagent (Sigma) and cleaned with peqGOLD phase-trapA (peqlab). RNA samples for RNA-seq were further purified with RNeasy Plant Mini Kit columns (Qiagen). For Northern hybridisation analyses, 10 µg of total RNA were separated in formaldehyde-containing agarose gels, blotted onto Hybond-N+ membranes (Amersham Biosciences), and hybridized with digoxigenin-labeled probes prepared as recommended by the manufacturer (Boehringer Mannheim). Templates for hybridization probes were generated by PCR amplification using oligonucleotides listed in Table S3. Autoradiographies were produced by exposing washed membranes to Fujifilm RX films. Binding of MetR to promoter regions of selected candidate targets was interrogated following the chromatin immunoprecipitation (ChIP) approach together with the GFP-Trap technology (ChromoTek) and using strain AfS171 that carries a codon-optimised version of the gfp2-5 allele [93] from pSK494 [94] preceded by a (GA) 5 linker region [95] fused C-terminally to the metR coding sequence. Essential steps were carried out following the protocol of [96] with modifications (see Supporting Information for details), enrichment for distinct fragments was probed by semi-quantitative PCRs with specific primer pairs covering respective promoter regions. Digital transcriptomes of A. fumigatus strains ATCC 46645 and AfS167 [metRΔ] were produced by Eurofins MWG Operon GmbH from two independent biological replicates, which underlay the high reproducibility of this experimental approach [97]. For this purpose, 3′-fragment-specific cDNA libraries were prepared from poly (A) -fragment selected mRNA and processed on the Illumina HiSeq 2000 sequencing system using v3. 0 chemistry and the 1×100 bp single read module, which ensures a high significance in respect to the copy number of each transcript. Mapping of reads on the most recent reference genome sequence (http: //www. aspergillusgenome. org/) was performed using BMA, SamTools and Picard software. To enable the direct comparison between the samples, the read count per reference has been normalized as follows: (total_mapped_reads_per_refseq/number_of_reads_in_sample) *lowest_total_sample_read_count and differential expression analyses were carried out using DESeq [98]. A 1. 5-fold change between the average number of reads in the wild-type and metRΔ strain was used as threshold to define genes which are expressed higher or lower, respectively, in the mutant. Functional characterization of these regulated genes was performed on the FungiFun website (https: //sbi. hki-jena. de/FungiFun/FungiFun. cgi) [55] based on both the FunCat method and the Gene Ontology (GO) classification [99]. Infections of larvae from the greater waxmoth Galleria mellonella were performed according to Kavanagh & Fallon [100]. Larvae were injected with varying doses of conidia in a saline solution supplemented with 0. 02% Tween 80 and 10 µl•ml−1 rifampicin, to avoid bacterial infections, and incubated at 30°C. Female mice (CD1 or BALB/c from Charles Rivers Breeding Laboratories, Sulzfeld, Germany) of 20 to 24 g were used for infection experiments. Immunosuppression was carried out by subcutaneous injection of 112 mg•kg−1 hydrocortisone acetate and intraperitoneal injection of 150 mg•kg−1 cyclophosphamide following a sequential protocol as previously described [101], with the modification that two doses of cortisone on days −3 and −1 were applied. Bacterial infections were prevented by adding 2 g•l−1 neomycin to the drinking water. Inocula were prepared by harvesting conidia from 5-day-old slants of solid medium followed by filtration through Miracloth tissue and washing with saline. Mice were anesthetized by intraperitoneal injection of a ketamine (1%) /xylazine (0,2%) solution and either infected intranasally by instillation of 2•105 conidiospores suspended in 40 µl of saline or intravenously by injection of a 50 µl suspension of 1•105 conidia into the lateral tail vein. Disease progression was followed twice daily by tabulating weight profiles and following the animals' behaviour. Signs of respiratory distress, hunched posture or poor mobility, as well as severe weight loss of more than 20% determined the experimental end point for each animal. To evaluate mortality rates in single-strain infection experiments, the log rank method was applied using the GraphPad Prism software. For competitive index (CI) assessment [13], mice were intranasally infected with a 1∶1 mixture of 2•104 conidia from ATCC 46645 and the metRΔ strain AfS167 and sacrificed after four days. The lungs were explanted and aliquots from homogenized tissue were spread onto media containing or lacking methionine as sulphur source to differentiate between the wild-type progenitor and its metRΔ deletant. The CI is defined as the output ratio of mutant to wild-type fungal colonies divided by the input ratio of mutant to wild-type fungal colonies [102]. Histological cryo-sectioning was performed on 4% formaldehyde-fixed lungs, staining procedures with hemotoxylin and eosin together with Grocott' s Methenamine Silver were carried out according to standard protocols. Five sections each from four lungs of mice infected with the wild-type and metRΔ strain, respectively, were inspected to yield representative images. Quantification of the intracellular siderophore ferricrocin (FC) was carried out as described earlier [40], [103]. Samples for total iron content measurements were lyophilized and 50 mg digested in 500 µl 60% HNO3 (Ultrapure, Merck) for 4 h at 110°C and diluted thereafter with ultrapure water (Milli-Q). Iron was quantified by graphite furnace atomic absorption spectrometry (M6 Zeeman GFAA-Spectrometer, Thermo Scientific) at 248. 3 nm and D2-Quadline background correction using 1000°C ash temperature and 2100°C atomization temperature under argon atmosphere. The iron content was calculated by interpolation from an appropriate standard curve (0. 5 to 12. 5 µg•l−1) using TraceCERT (Sigma-Aldrich) standard solution. The accuracy of analysis was assessed by simultaneous analysis of a standard reference human serum sample (ClinChek, Recipe).
Invasive pulmonary aspergillosis (IPA) is a life-threatening disease that affects primarily immunosuppressed patients. During the last decades the incidence of this disease that is accompanied by high mortality rates has increased. Since opportunistic pathogenic fungi, unlike other pathogens, do not express specific virulence factors, it is becoming more and more clear that the elucidation of fungal metabolism is an essential task to understand fungal pathogenicity and to identify novel antifungal targets. In this work we report genetic inactivation of the sulphur transcription regulator MetR in Aspergillus fumigatus and subsequent study of the resulting phenotypes and transcriptional deregulation of the mutant. Here we show that regulation of sulphur assimilation is an essential process for the manifestation of IPA. Moreover, a regulatory connection between sulphur metabolism and iron homeostasis, a further essential virulence determinant of A. fumigatus, is demonstrated in this study for the first time. A deeper knowledge of sulphur metabolism holds the promise of increasing our understanding of fungal virulence and might lead to improved antifungal therapy.
microbial metabolism
mycology
medical microbiology
biology
microbiology
pathogenesis
fungal physiology
Regulation of Sulphur Assimilation Is Essential for Virulence and Affects Iron Homeostasis of the Human-Pathogenic Mould Aspergillus fumigatus