Mitogen-Activated Protein Kinase Cross-Talk Interaction Modulates the Production of Melanins in Aspergillus fumigatus.

The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade.IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.

released into the air and, when inhaled by immunocompromised patients, can cause severe diseases, including invasive aspergillosis (IA). An increase in the incidence of IA has been observed in the last decades, and the mortality attributed to IA infections can reach 90%. IA is a multifactorial disease, and A. fumigatus has several phenotypic characteristics that make it an aggressive opportunistic pathogen (2). Several factors contribute to A. fumigatus virulence, such as production of dihydroxynaphthalene (DHN)-melanin, hypoxia resistance, ability to subtract environmental iron, toxin production, thermotolerance, and distinct surface molecules (3)(4)(5)(6)(7).
Mitogen-activated protein kinase (MAPK) pathways are important for the transmission, integration, and amplification of signals and are essential components involved in diverse cellular processes in eukaryotes (8). In fungi, MAPK pathways regulate cellular responses to different kinds of stresses (9)(10)(11). The central module of each MAPK signaling pathway consists of three protein kinases: a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAPK. The MAPK cascades are normally triggered by upstream sensors (e.g., receptors) and end with the activation of downstream elements, such as transcriptional regulators (12).
MAPK signaling cascades have been well characterized in yeasts (13)(14)(15)(16). In filamentous fungi, their function was mainly assigned to pheromone responses and filamentous growth, osmotic stress, and cell wall integrity. Additionally, it was demonstrated that MAPKs influence several phenotypes relevant for pathogenesis in both human and plant pathogens (9,11).
A. fumigatus contains four MAPKs: MpkA, which mainly regulates cell wall integrity (17); MpkC and SakA, similar to the Saccharomyces cerevisiae Hog1, which are involved in the response to oxidative stress and in polyalcohol sugar metabolism (18)(19)(20); and MpkB, homologous to yeast Fus3, thus far uncharacterized (21). A cross talk interaction between SakA and MpkA has already been observed for the adaptation to the antimycotic drug caspofungin (22,23). In addition, it was shown that MpkC and SakA cooperate in the responses to different types of stresses in A. fumigatus, including the response to osmotic and oxidative stress, high-temperature adaptation, cell wall damage, and virulence (23).
Previous investigations on Fus3/MpkB orthologs in several phytopathogenic fungi showed their conserved important role in plant infection (11). In parallel, in other important human pathogenic fungi, such as Candida albicans and Cryptococcus neoformans, the deletion of their FUS3 orthologs mainly caused the loss of mating efficiency and decrease of biofilm formation (24,25). Of note, in the closely related species Aspergillus nidulans, the mpkB deletion not only inhibited sexual crossing, as expected, but also affected the production of relevant secondary metabolites, such as the mycotoxin sterigmatocystin, the antitumor agent terrequinone A, and penicillin (26,27).
In the present study, we set out to analyze the function of the MpkB regulatory network in A. fumigatus. Deletion of mpkB increased sensitivity to the glucan synthase inhibitor caspofungin, suggesting a role in cell wall biosynthesis. Furthermore, we observed an increase of dark pigments during growth in liquid medium that was related to DHN-melanin production. Melanins are a class of dark-brown pigments often associated with the cell wall. Their main role is to protect the organisms from exogenous stressors, thereby contributing to the first line of defense against external hazards (7,28). A. fumigatus produces two types of melanins: pyomelanin, which derives from the catabolism of tyrosine via the intermediate homogentisate, and DHN-melanin, which is produced as a polyketide derivative and is responsible for the gray-green color of the spores (7). Additionally, DHN-melanin plays a crucial role in conidial immune evasion and, consequently, fungal virulence (29)(30)(31). In the A. fumigatus genome, the genes coding for DHN-melanin biosynthetic enzymes, as well as those for pyomelanin, are clustered (32,33).
Signaling pathways that regulate the expression of melanin biosynthesis genes are barely known. DHN-melanin was found to be regulated by cAMP signaling, and in A. fumigatus, two transcription factors responsible for its regulation were identified: DevR, a basic helix-loop-helix (bHLH) transcription factor, and RlmA, a MADS-box transcription factor actively involved in cell wall integrity (34,35). The production of pyomelanin was also associated with cell wall stress, highlighting its function as a protective compound (36). Highly important here, we observed that the deletion of the gene coding for the G␣ protein GpaA and the gene coding for the G protein-coupled receptor GprM resulted in phenotypes similar to that resulting from mpkB deletion, suggesting that they are involved in the same signaling pathway. Here, we present a detailed characterization of the MAPK MpkB in A. fumigatus that led to the identification of its physiological role and several components of its regulatory circuits.
in the wild-type strain (Fig. 2D). These results indicate that MpkB plays an important role in the prevention of conidium formation during active vegetative growth.
G␣ protein GpaA and G protein-coupled receptor GprM are important for repressing DHN-melanin production. It has been previously reported that pksP transcription is connected to cAMP signaling via protein kinase A (PKA) (34). In order to identify further pathways involved in DHN-melanin regulation, we exploited the darkcolor production as a readout system to identify additional signal proteins. Previous studies reported that the deletion of gpaB, coding for a G␣ protein involved in the cAMP/PKA pathway, strongly decreased DHN-melanin production (39). Besides GpaB, A. fumigatus has two additional G␣ proteins, GpaA and GpaC (40). Phenotypic analysis of mutant strains with deletions of the three G␣ proteins revealed that only the ΔgpaA tests were performed by inoculating A. fumigatus conidia onto solid MM agar plates with or without sorbitol. Agar plates were incubated for 5 days at 37°C. (B) Numbers of conidia produced in the wild-type, ΔmpkB, and mpkBc (complemented) strains. The results are the averages of three repetitions Ϯ standard deviations. One-way analysis of variance (ANOVA) with Tukey's multiple comparison test was used for statistical analysis, and an asterisk represents a P value of Ͻ0.05. (C) A. fumigatus conidia were inoculated into liquid MM for 72 h at 37°C with 200 rpm shaking. (D) Results of qRT-PCRs showing the A. fumigatus pksP mRNA steady-state levels in the wild-type and ΔmpkB strains grown at 37°C for 24 and 48 h, respectively. The results represent the averages of three repetitions Ϯ standard deviations and are expressed as the cDNA concentration of pksP divided by the cDNA concentration of the ␤-tubulin gene. The results are the averages of three repetitions Ϯ standard deviation. One-way ANOVA with Bonferroni posttest was used for statistical analysis, and an asterisk represents a P value of Ͻ0.05. (E) Visualization of melanin by copper-silver staining of hyphae and conidiophores of wild-type and ΔmpkB strains grown for 48 h at 37°C. Bars, 10 m. mutant showed increased pigment production in submerged culture (Fig. 3A). Parallel deletion of the pksP gene in the ΔgpaA mutant background confirmed that, in this strain also, the dark pigment was DHN-melanin ( Fig. 3A).
In eukaryotes, G␣ proteins are normally associated with receptors, and in particular G protein-coupled receptors (GPCRs). We screened a comprehensive collection that included 12 A. fumigatus GPCR null mutants (Table 1). Spores for each mutant were inoculated into liquid medium at 37°C for 3 days with shaking (data not shown). Among the screened mutants, only the ΔgprM mutant produced more dark pigments than the wild type, and in this case also, ΔgprM complementation and the parallel deletion of the pksP gene confirmed that the phenotype was connected to DHN-melanin (Fig. 3A).
In order to check whether the GprM receptor and GpaA interact directly, we employed a split-ubiquitin yeast assay two-hybrid screen with A. fumigatus GprM as bait and the three G␣ proteins as preys. This experiment clearly demonstrated that among the G␣ proteins expressed by A. fumigatus, GprM interacts exclusively with GpaA (Fig. 3B). Two other yeast proteins, S. cerevisiae Gap1 and Sui2, previously shown to interact by using a split-ubiquitin yeast assay two-hybrid screen, were used as positive controls (41).
Finally, the interaction between the GprM receptor and GpaA was validated in A. fumigatus. We expressed a green fluorescent protein (GFP)-tagged gpaA gene with a hemagglutinin (HA)-tagged gprM as a dicistronic gene. The two fragments were spaced by the 2A peptide sequence and then placed downstream from a tetracycline-inducible promoter, Tet ON , optimized for fungal expression (42,43). The plasmid obtained was used to transform A. fumigatus strain CEA10 by ectopic integration. By using anti-GFP antibody for the coimmunoprecipitation (co-IP) experiment, we could also isolate the expressed GprM-3ϫHA receptor (Fig. 3C, left). The opposite experiment, co-IP using the anti-HA antibody, gave the same results, with the GpaA-GFP protein identified by Western blotting (Fig. 3C, right). It is worth noticing that the anti-GFP antibody also recognized unspecific bands (ϳ50 kDa) from the beads; however, because of the difference in size compared to that of the GpaA-GFP protein, a clear band was still discernible.
Cross-talk interaction between the MpkB and MpkA pathways. The screening conducted against the different cell wall-acting compounds revealed that the ΔmpkB mutant is more sensitive to the ␤-1,3-glucan synthase inhibitor caspofungin, with significant reduction of growth when compared to that of the recipient strain ( Fig. 4A and B and Fig. S1). The ΔmpkB mutant had also lost the caspofungin paradoxical effect, fumigatus GprM as the bait and the three G␣ proteins as preys. Two other yeast proteins, Gap1 and Sui2, previously shown as interacting by using a split-ubiquitin yeast two-hybrid screen, were used as positive controls (41). (C) Coimmunoprecipitation (co-IP) was performed with total protein extracted from the A. fumigatus wild-type strain (lanes 1 and 4), A. fumigatus expressing a dicistronic gene including the gfp-and the HA-tagged gprM (lanes 2 and 5), and A. fumigatus expressing a dicistronic gene including the gfp-tagged gpaA and the HA-tagged gprM (lanes 3 and 6). Co-IP performed with an anti-GFP antibody specifically isolates the GprM-3ϫHA protein only in the presence of the GFP-tagged GpaA (left, lane 3). Similarly, co-IP performed using an anti-HA antibody specifically isolates the GpaA-GFP protein, but not GFP alone (right, lane 3). The experiment also shows that the anti-GFP antibody used recognizes an unspecific band detectable also when the beads are not attached to any protein (right, lane B). However, because of the difference in size, a clear band showing the expressed GpaA-GFP protein was detected (right, lane 3). Lanes M, SeeBlue plus2 marker (ThermoFisher). a phenomenon where high caspofungin concentrations revert the anticipated inhibition of A. fumigatus growth ( Fig. 4A and B) (44). This sensitivity was almost comparable with that observed for the ΔmpkA mutant; MpkA is the second p42/44 kinase encoded by A. fumigatus and mainly responsible for cell wall maintenance under stress conditions (45). Concerning MpkA, previously published transcriptomic studies reported that this kinase positively affects the expression of the DHN-melanin gene cluster (22,46). These observations had not yet been proved by ad hoc experiments. We constructed an ΔmpkA ΔmpkB double deletion strain aiming to investigate possible interactions between the two kinases. As shown by the results in Fig. 4C, pigmentation of the medium was not observed for either the ΔmpkA or the ΔmpkA ΔmpkB mutant, suggesting that MpkA positively regulates DHN-melanin biosynthesis while MpkB negatively affects its production.
To investigate whether MpkB repression affects MpkA activation, we checked the MpkA phosphorylation levels in the mutants by immunoblot assay. We detected higher MpkA phosphorylation during the first 24 h of incubation for the ΔmpkB and ΔgpaA strains than for the wild type, and in the ΔgprM mutant, MpkA was shown to be highly activated even 48 h after incubation (Fig. 4D). These data confirmed that GpaA and GprM act upstream from MpkB and that the inhibition of this pathway increased MpkA phosphorylation, leading to higher DHN-melanin accumulation. The mechanism is still unknown; however, an indirect interaction is most likely, since binding between MpkA and MpkB was not detected by a coimmunoprecipitation assay performed with the two kinases (Fig. S3). Transmission electron microscopy (TEM) analysis showed that untreated ΔmpkB germlings (10 and 24 h) grown in MM had approximately 2-fold-thicker cell walls than the wild type ( Fig. 5A and B). Cell wall stains and lectins were used to identify differences in the content or exposure of different carbohydrates on the surface of the fungal cell wall in both the wild type and the ΔmpkB mutant. These included the following: (i) soybean agglutinin (SBA)-fluorescein isothiocyanate (FITC) (preferentially binds to oligosaccharide structures with terminal ␣or ␤-linked N-acetylgalactosamine [GalNAc] and, to a lesser extent, galactose residues, which are important for recognizing galactosaminogalactan [GAG]); (ii) wheat germ agglutinin (WGA)-FITC (recognizing surface-exposed glucosamine [Glc]); (iii) ConA (concanavalin A)-FITC (recognizes ␣-linked mannose); (iv) soluble dectin-1 stain (recognizing ␤-glucans); and (v) CFW (recognizing chitin). There were no differences in the levels of ␤-1,3-glucan, chitin, or N-acetylgalactosamine in the wild-type, ΔmpkB, and complemented strains (Fig. 5C to E and G). In contrast, the ΔmpkB mutant had about 3-fold-less glucosamine and a lower level of ␣-linked mannose than the wild-type strain (Fig. 5F).
Finally, we investigated whether the misbalance in MpkA phosphorylation caused any effect on the production of pyomelanin. As previously reported, the cell wall stress induced by the deletion of mpkA promotes the formation of pyomelanin as a compensatory response (36). The increase in pyomelanin formation can be detected by testing the activity of the homogentisate 1,2-dioxygenase (HmgA) involved in tyrosine degradation (32). After adding L-tyrosine to the medium, a reduction in HmgA activity induced higher pyomelanin accumulation, and vice versa (Fig. 6A). By growing the mutants in liquid MM plus L-tyrosine for 24 h, we could not detect a significant change in HmgA activity for the mpkA deletion mutants. However, impairing MpkB signaling resulted in higher HmgA enzymatic activities for the ΔgprM, ΔgpaA, and ΔmpkB deletion mutants (Fig. 6B). This effect was conserved during the incubation time and was still detectable after 48 h. The mutant with the deletion of mpkA showed an increase of HmgA activity after 48 h of incubation, which is not in line with our previous results (36). For comparison to the results of the earlier activity test, we performed the HmgA activity test using smaller volumes (each sample had a total volume of 100 l and was disposed in a 96-well plate) and reading the absorbance using a plate reader. However, aside from this incongruence, the double deletion of mpkA and mpkB restored the HmgA activity level, confirming the involvement of the cell wall integrity pathway in pyomelanin formation and highlighting once again the conflicting roles of the MpkA and MpkB pathways.
Cell wall stress enhances MpkB nuclear localization. To assess the involvement and subcellular localization of MpkB during growth and under stress conditions, we generated a strain expressing MpkB-GFP under the control of its own endogenous Statistical analysis was performed using the one-tailed, paired t test, comparing data to the results for the control condition (*, P Ͻ 0.00001). (C to G) Detection of different sugars exposed on the cell surface. Conidia were cultured in liquid MM to the hyphal stage, UV killed, and stained with CFW, soluble dectin-1, or specific probe to detect the content of exposed sugars. Experiments were performed in triplicate, and the results are displayed as mean values with standard errors (two-way ANOVA followed by Tukey's; *, P Ͻ 0.05).
promoter. This was achieved by replacing the wild-type allele with the mpkB-gfp cassette; the strain generated behaved identically to the wild-type strain (Fig. S3). When the MpkB-GFP strain was grown in MM for 16 h at 30°C, a very weak and diffuse fluorescent signal was observed in the cytosol and about 1% of the germlings' stained nuclei overlapped with the GFP signal (Fig. 7A). The increase of MpkB nuclear localization was observed upon mpkA deletion, and this condition was similar when the growing mycelium was challenged with the osmotic stress inducer sorbitol. Moreover, the exposure to different caspofungin concentrations increased nuclear localization over time, with MpkB localized in almost one-third of the germlings 30 min after incubation. As for sorbitol, this phenomenon was strongly enhanced by mpkA deletion. These data confirmed that MpkB can activate compensatory pathways in the absence of MpkA during cell wall stress.
Cell wall-impaired mutants are normally sensitive to heat shock (36). For the ΔmpkB strain, incubation at high temperatures affected the sporulation ability more than it did radial growth (Fig. 7B). MpkB nuclear localization was increased by incubating A. fumigatus at 44°C (Fig. 7A); however, the parallel deletion of mpkA did not affect MpkB activation, demonstrating that the interaction of the two pathways is stress related. This ΔmpkB mutant strains were assayed for HmgA activity. After 18 h of growth, 10 mM L-tyrosine was added to the culture. Samples were harvested, and the protein extracts were incubated with homogentisate for 35 min. Formation of maleylacetoacetate was monitored at 330 nm. Total protein extracted from the ΔhmgA and ΔhppD mutant strains was used as the negative and positive control, respectively (data not shown). HmgA activity was determined in protein extracts of mycelia harvested after 24 and 48 h postinoculation (average value Ϯ standard deviation). Statistical significance was determined for the results of all of the experiments by Student's t test. Significance of differences of data in comparison to the results for the wild-type strain is indicated as follows: *, P Ͻ 0.1; **, P Ͻ 0.01. was even clearer when A. fumigatus was challenged with H 2 O 2 . As previously reported, hydrogen peroxide strongly induces MpkA activation, and the ΔmpkA mutant is very susceptible to oxidative stress (47). Here, we observed that H 2 O 2 enhanced MpkB localization to the nucleus as well, but the lack of mpkA significantly decreased MpkB nuclear transport. It is possible that an additional salvage pathway that is not activated by MpkB operates during oxidative stress. This observation is supported by the fact that mpkB deletion did not affect H 2 O 2 sensitivity (Fig. S2), suggesting that during oxidative stress, MpkB is transported into the nuclei but is likely still inactive.
A. fumigatus ⌬mpkB is still virulent in a low-dose murine infection model. In order to determine whether the lack of mpkB affects A. fumigatus virulence, the ΔmpkB strain, the mpkBc (complemented) strain, and the respective wild-type strain were tested in a murine infection model of IA (Fig. S4). The results of this experiment indicated that mpkB is not essential for virulence of A. fumigatus.
Ethics statement. The principles that guide our studies are based on the Declaration of Animal Rights ratified by the UNESCO in January 27, 1978, in its eighth and 14th articles. All protocols used in this study were approved by the local ethics committee for animal experiments from the Campus of Ribeirão Preto, Universidade de São Paulo (Permit Number: 08.1.1277.53.6; Studies on the interaction of A. fumigatus with animals). All animals were housed in groups of five within individually ventilated cages and were cared for in strict accordance with the principles outlined by the Brazilian College of Animal Experimentation (Princípios Eticos na Experimentacão Animal-Colégio Brasileiro de Experimentacão Animal, COBEA) and Guiding Principles for Research Involving Animals and Human Beings, American Physiological Society. All efforts were made to minimize suffering. Animals were clinically monitored at least twice daily and humanely sacrificed if moribund (defined by lethargy, dyspnea, hypothermia, and weight loss). All stressed animals were sacrificed by cervical dislocation.

DISCUSSION
A number of studies have characterized MAPKs with a focus on their role in pathogenesis and secondary metabolite production (9, 10, 48, 49). The pathogenic MAP Kinases and Melanin Production in A. fumigatus ® fungus A. fumigatus codes for four MAPKs, and until now, MpkB was the only one still uncharacterized. As shown by the experiments whose results are described here, the deletion of the A. fumigatus mpkB gene reduced conidium production on solid medium but induced the formation of conidiophores in submerged cultures. In A. nidulans, MpkB was found to be essential for sexual development, and the ΔmpkB mutant produced conidia in submerged cultures with constant brlA mRNA accumulation (50,51). We also observed increased accumulation ofbrlA during growth of the A. fumigatus ΔmpkB mutant in submerged cultures, indicating that in both fungal systems, MpkB is important for temporal brlA expression. However, other than its role in spore development, MpkB had no impact on A. fumigatus virulence, since the ΔmpkB mutant showed wild-type-like virulence in a murine infection model. Previously, Atoui et al. (26) have shown that in the A. nidulans ΔmpkB mutant, the expression of genes involved in sterigmatocystin, penicillin, and terrequinone A biosynthesis, as well as the expression of laeA (loss of aflatoxin expression A, encodes global regulator of secondary metabolism), were significantly reduced (26). Similarly, MpkB also affects secondary metabolite production in A. fumigatus, in particular DHN-melanin, but surprisingly, does not confer resistance to oxidative stress agents. As shown herein, the MpkB-GFP translocation to the nucleus is affected by the presence of MpkA during cell wall stress. The negative interaction between MpkA and MpkB seems to be mutual in particular during cell wall stress; indeed, the deletion of mpkB increased MpkA phosphorylation, while the deletion of mpkA increased MpkB nuclear localization. Since MpkA phosphorylation was increased in the ΔmpkB mutant, the higher melanin production seems to be related to aberrant MpkA activation.
The involvement of the cell wall integrity signaling pathway in melanin production has already been hypothesized. The analysis of transcriptome data of ΔmpkA mutants and the involvement of the cell wall-related transcription factor RlmA in the DHNmelanin gene cluster activation already suggested that, besides the cAMP pathway, additional signal pathways are involved in melanin regulation (35,46,52). Taking into account that melanin production is lower in the ΔmpkA ΔmpkB double mutant than in the single mpkB deletion strain, this finding supports a model in which the expression of the DHN-melanin biosynthesis genes is controlled by the RlmA transcription factor (Fig. 8).
Thus far, the cAMP/PKA pathway was the only regulatory signaling cascade associated with DHN-melanin production in A. fumigatus (34,53). Null mutants with deletions of genes involved in this pathway, such as the G protein ␣ subunit GpaB and adenylate cyclase AcyA, showed reductions in DHN-melanin production, while overexpression of the gene encoding the protein kinase A catalytic subunit 1 (PkaC1) induced the expression of the DHN-melanin cluster genes (Fig. 8) (27). This was also observed in other pathogenic fungi, such as C. neoformans, where mutations in the G protein Gpa1, adenylate cyclase Cac1, and/or the catalytic subunit of PKA Pka1 reduced the formation of melanins (54,55).
Here, we report that an additional G protein ␣ subunit (GpaA) and the GPCR GprM are also involved in the regulation of DHN-melanin production (Fig. 8). Accordingly, GprM physically interacts with GpaA, suggesting that melanin production in A. fumigatus is also mediated via the GprM/GpaA/MpkB pathway. Genome-wide analysis revealed that the A. fumigatus genome codes for three G protein ␣ subunits, one G-␤, and one G-␥, while it codes for at least 15 different GPCRs (53,56). So far, gprD and gprC have been characterized in A. fumigatus, and their role was connected to hyphal morphogenesis and virulence (57). The high number of GPCRs encoded by fungal genomes makes their study difficult, because the majority of GPCR gene single deletion mutants do not show a discernible phenotype (data not shown). This was also experienced by studying a comprehensive Aspergillus flavus GPCR mutant library, where only a few mutants showed growth defects or impaired toxin production (58). Therefore, until now, there have only been a few examples of filamentous fungi for which the characterization of a signal pathway from the receptor to the activation of response genes has been reported.
In conclusion, here we report the first characterization of MpkB in A. fumigatus, showing its role in conidium formation and development and in the regulation of DHN-melanin production. Additionally, we demonstrate that MpkB interacts with the MpkA signaling pathway, particularly during the caspofungin stress response and melanin production. How this global cross talk affects transcriptional regulators needs to be elucidated, but in the future, the expression of DHN-melanin biosynthesis genes, with its known transcription factors, can be used to address these questions.

MATERIALS AND METHODS
Strains and media. The A. fumigatus strains used in this study are described in Table 1. Detailed information about plasmids and the generation of mutant strains is reported in Text S1 and Fig. S5 in the supplemental material. Complete media were of the following two basic types: a complete yeast extract-glucose (YAG) medium (2% [wt/vol] glucose, 0.5% [wt/vol] yeast extract, 2% [wt/vol] agar, trace elements) with three variants (a YUU medium [YAG medium supplemented with 1.2 g/liter each of uracil and uridine] and liquid YG or YUU medium of the same composition but without agar) and a minimal medium (MM; 1% [wt/vol] glucose, original high nitrate salts, trace elements, 2% [wt/vol] agar, pH 6.5). The trace elements, vitamins, and nitrate salts used were described previously (59). S. cerevisiae strains were grown in synthetic dextrose (SD) medium (6.7 g Difco yeast nitrogen base [YNB] without amino acids, with distilled water to 950 ml, 20 g agar, and 50 ml sterile 40% [wt/vol] glucose).
Conidium production. Five microliters of 2 ϫ 10 7 conidia of each strain was overlaid onto MM agar plates, followed by incubation at 37°C for 5 days. Fresh conidia were harvested in 10 ml of 0.01% (vol/vol) Tween 80, vortexed for 30 s, and filtered through miracloth. Conidial suspensions were spun for 5 min at 3,000 ϫ g, and pellets were resuspended using 10 ml H 2 O. The numbers of conidia were counted using a hemacytometer. This experiment was performed in triplicate.
Phenotypic assays. The phenotypes of the deletion mutants were evaluated either by measuring radial growth or by assessing the initial growth of a droplet of conidia from a serial dilution at different temperatures and in the presence or absence of oxidative and osmotic stressing agents. Dropout  (39,53). The GPCR GprM, which interacts with GpaA, activates the MpkB pathway. MpkB activation affects MpkA, which regulates the DHN-melanin gene cluster. Finally, the transcription factor RlmA directly regulates the expression of the melanin genes by directly binding the pksP promoter (35). Parallel to the expression of the DHN-melanin gene cluster, the activation of MpkB influences the HmgA activity, promoting the accumulation of pyomelanin. ACYA, adenylate cyclase; PKAR, PKA regulatory subunit. experiments were performed using 5-l amounts of a 10-fold dilution series starting at a concentration of 2 ϫ 10 7 for the wild-type and mutant strains spotted on different growth media and grown for 48 h at 37°C. The A. fumigatus crystal violet assay was performed as previously described (60,61). All the experiments were performed at least three times. We also screened a collection of 12 GPCR mutants (Table 1) for the production of dark pigments by growing them in liquid MM for 48 h at 37°C.
Split-ubiquitin yeast two-hybrid assay. The A. fumigatus gprM, gpaA, gpaB, and gpaC open reading frames were synthesized and cloned into the pTMBV4 and pDL2XN plasmids, respectively (41). The genes of interest were expressed in Saccharomyces cerevisiae strain NMY32. The NMY32 strain was initially transformed with the gprM-bearing plasmid and later with one of the gpaA-, gpaB-, or gpaC-bearing plasmids. Then, we used these transformants for the split-ubiquitin membrane-based yeast two-hybrid assay. First, these strains were inoculated onto SD-Leu-Trp selective culture medium and grown overnight. The absorbance of the inoculum was measured through spectrophotometry, and later, all samples were standardized to an optical density at 600 nm (OD 600 ) of 1. Tenfold serial dilutions were made, and these dilutions were spotted on agar plates with different selective culture media, including SD-Leu-Trp plus X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside), SD-Leu-Trp-Ade, SD-Leu-Trp-His, SD-Leu-Trp-His plus 3-AT (3-amino-1,2,4-triazole), or SD-Leu-Trp-Ade-His medium. The agar plates were incubated for 3 days at 30°C.
Staining and microscopy. The copper-silver staining experiments were performed by a modification of a previously described procedure (37). Briefly, the wild-type and ΔmpkB mutant strains were grown on solid MM on slides. After growth, the slide cultures were incubated in 10 mM copper sulfate solution at room temperature for 1 h and, after washing, treated with 1.0% (wt/vol) sodium sulfide solution for 1 h in the dark. Subsequently, the slide cultures were incubated for 4 min in a solution of 22 mg silver lactate and 170 mg hydroquinone (HQ) in a citrate buffer (0.1 M, pH 3.7) solution (Ag-HQ solution) at room temperature. The copper and sulfide treatments were followed by repeated washings (six times with 2 ml distilled water). Afterward, slides were visualized on a Zeiss Axio Observer Z1 fluorescence microscope. Bright-field images were captured with an AxioCam camera (Carl Zeiss) and processed using AxioVision software (version 4.8). For fluorescence experiments, MpkB-GFP strain conidia were cultivated on coverslips in 4 ml of YG medium for 16 h at 30°C. After incubation, subsets of coverslips with adherent germlings were left untreated or treated with 1.0 M sorbitol, different concentrations of H 2 O 2 , or 0.125 g/ml caspofungin for different periods of time. Subsequently, the coverslips were rinsed with phosphate-buffered saline (PBS; 140 mM NaCl, 2 mM KCl, 10 mM NaHPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) and incubated for 3 min in a solution with Hoechst stain (Life Technologies) (12 g/ml). After incubation with the dye, the coverslips were washed with PBS and mounted for examination. Slides were visualized on an Observer Z1 fluorescence microscope using a 100ϫ objective oil immersion lens (for GFP, filter set 38 high efficiency [HE], excitation wavelengths of 450 to 490 nm, and emission wavelengths of 525/50 nm; for Hoechst stain, filter set 49, excitation wavelengths of 365 nm, and emission wavelengths of 420 to 470 nm). Differential interference contrast (DIC) images and fluorescence images were captured with an AxioCam camera and processed using AxioVision software (version 4.8).
Homogentisate dioxygenase assay. A. fumigatus was grown in liquid MM at 37°C and 180 rpm shaking for 18 h. Afterwards, 10 mM L-tyrosine was added. Samples for the homogentisate dioxygenase assay were collected 6 and 30 h after induction (in total, 24 and 48 h of growth, respectively). The mycelia were harvested, washed with water, frozen in liquid nitrogen, and stored at Ϫ80°C overnight. For protein preparation, the mycelia were powdered in liquid nitrogen and resuspended in 50 mM KPO 4 , pH 7.0. After centrifugation at 16,000 ϫ g, 4°C, for 15 min, the supernatants were collected and the protein concentrations were determined using the Coomassie plus protein assay (ThermoFisher Scientific) according to the manufacturer's instructions. The homogentisate dioxygenase assay was performed as described previously (32), in a 9-well plate in a total volume of 100 l with 2 g of total protein for each sample. All measurements were done with three biological and three technical replicates. Proteins extracted from the A. fumigatus ΔhmgA and ΔhppD mutants were used as negative and positive controls, respectively (not shown) (32). All the measurements were performed using a ClarioSTAR plate reader (BMG Labtech).
Coimmunoprecipitation of GpaA and GprM. For co-IP studies of GpaA and GprM, C-terminally tagged GpaA-GFP and GprM-3ϫHA and GFP alone were constructed and gpaA-GFP::gprM-3ϫHA and GFP::gprM-3ϫHA strains generated. Amounts of 50 ml of MM were inoculated with 1 ϫ 10 7 spores of the two mutant strains and the wild type and incubated at 37°C with shaking. After 16 h, 20 g/ml doxycycline was added. Mycelia were harvested after an additional 4 h of incubation, flash-frozen, and stored at Ϫ80°C. For protein preparation, the mycelia were powdered in liquid nitrogen and resuspended in 500 l Tris buffer (0.5 M NaCl, 0.1 M Tris, pH 8.0) including EDTA-free protease inhibitor cocktail (Roche), 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], and 1 mM EDTA. Samples were vortexed 3ϫ for 10 s and afterwards centrifuged for 20 min at 4°C and 16,000 ϫ g. The supernatant was removed, and protein concentration was determined using the Bradford assay (Pierce Coomassie-plus; Fisher Scientific). For reciprocal co-IP, protein A Dynabeads (Thermofisher Scientific) were incubated with anti-3ϫHA (Sigma) or anti-GFP B2 (SantaCruz Biotechnology) antibody for 30 min at room temperature. Before adding 100 g of protein solution to 80 l of beads for 3 h while shaking at 8°C, the beads were washed three times with resuspension buffer. After incubation, the beads were washed three times with resuspension buffer and resuspended in 20 l buffer, and bound proteins were eluted by adding sample buffer and boiling at 95°C for 5 min. Afterwards, proteins were separated on X10 Bolt 4 to 12% Bis-Tris plus gels (Fisher Scientific) and transferred to a nitrocellulose membrane using an iBlot2 blotting system (ThermoFisher Scientific). GFP and GFP-tagged GpaA were detected using an HRP-linked anti-GFP B2 antibody (Santa Cruz Biotechnology) diluted 1:2,500 in blocking solution (5% [wt/vol] milk powder in TBST). 3ϫHA-tagged GprM was detected using the antibodies and dilutions described above. The antibodies were diluted in blocking solution. For controls for the anti-GFP antibody blotting, 20 g of wild-type and GpaA-GFP::GprM-3ϫHA and 1 g of GFP::GprM-3ϫHA protein preparations were used. For controls for the anti-3ϫHA antibody blotting, 2 g of all protein preparations were used. Detection was performed using WesternSure premium chemiluminescent substrate (Li-Cor GmbH) and a Fusion system (Vilber Lourmat).