The capacity of Aspergillus niger to sense and respond to cell wall stress requires at least three transcription factors: RlmA, MsnA and CrzA
© Fiedler et al.; licensee BioMed Central Ltd. 2014
Received: 2 April 2014
Accepted: 18 August 2014
Published: 1 December 2014
Cell wall integrity, vesicle transport and protein secretion are key factors contributing to the vitality and productivity of filamentous fungal cell factories such as Aspergillus niger. In order to pioneer rational strain improvement programs, fundamental knowledge on the genetic basis of these processes is required. The aim of the present study was thus to unravel survival strategies of A. niger when challenged with compounds interfering directly or indirectly with its cell wall integrity: calcofluor white, caspofungin, aureobasidin A, FK506 and fenpropimorph.
Transcriptomics signatures of A. niger and phenotypic analyses of selected null mutant strains were used to predict regulator proteins mediating the survival responses against these stressors. This integrated approach allowed us to reconstruct a model for the cell wall salvage gene network of A. niger that ensures survival of the fungus upon cell surface stress. The model predicts that (i) caspofungin and aureobasidin A induce the cell wall integrity pathway as a main compensatory response via induction of RhoB and RhoD, respectively, eventually activating the mitogen-activated protein kinase kinase MkkA and the transcription factor RlmA. (ii) RlmA is the main transcription factor required for the protection against calcofluor white but it cooperates with MsnA and CrzA to ensure survival of A. niger when challenged with caspofungin and aureobasidin A. (iii) Membrane stress provoked by aureobasidin A via disturbance of sphingolipid synthesis induces cell wall stress, whereas fenpropimorph-induced disturbance of ergosterol synthesis does not.
The present work uncovered a sophisticated defence system of A. niger which employs at least three transcription factors - RlmA, MsnA and CrzA – to protect itself against cell wall stress. The transcriptomic data furthermore predicts a fourth transfactor, SrbA, which seems to be specifically important to survive fenpropimorph-induced cell membrane stress. Future studies will disclose how these regulators are interlocked in different signaling pathways to secure survival of A. niger under different cell wall stress conditions.
KeywordsCell signaling Cell wall Chitin Drug action Fungi Transcriptomics
Fungi are among the most serious biological threats to humans and plants. They kill as many people as tuberculosis and malaria and spoil about 10% of the world’s annual harvest ,. Although several antifungal drugs are available, their success is limited due to toxicity, a narrow spectrum of activity, detrimental drug interactions and the development of drug resistance . To mitigate this public threat, safe and effective antifungal drugs are therefore needed. Key to this is a better understanding on how fungi sense and respond to antifungal drugs.
A preferred target for new antifungal drugs is the fungal cell wall as its composition is fundamentally different from bacterial and plant cell walls. In addition, the cell wall from yeast and filamentous fungi display significant architectural differences which can potentially be exploited. Whereas β-1,3-glucans, β-1,4 glucans, β-1,6-glucans, chitin, chitosan and glycoproteins are major constituents found in both, α-1,3-glucans and galactomannans are specific for filamentous fungi. Furthermore, chitin is much more abundant in cell walls of filamentous fungi -. At least three signaling pathways have been shown to be involved in the cell wall compensatory response in the yeast model Saccharomyces cerevisiae when confronted with cell wall disturbing compounds: the Pkc1p-Slt2p signaling pathway (also named cell wall integrity (CWI) pathway) mediated by the transcription factors Rlm1p and Swi4p/Swi6p, the general stress response pathway mediated by Msn2p/Msn4p, and the calcium/calcineurin pathway mediated by Crz1p ,. Whereas cell wall stress responses are well studied and understood in S. cerevisiae, much less is known from Aspergilli, a genus comprising many human and plant pathogenic filamentous fungi. Components of the CWI signaling cascade, the general stress response pathway and the calcium/calcineurin pathway are, however, conserved among Aspergilli -.
Using A. niger as a model system, we recently studied its defense strategies against cell-surface acting compounds such as caspofungin (CA, inhibitor of β-1,3-glucan synthesis), fenpropimorph (FP, inhibitor of ergosterol synthesis), the antifungal protein AFP (inhibitor of chitin synthesis) and calcofluor white (CFW, inhibitor of chitin microfibril assembly) ,,. Common to these compounds is that they induce the CWI pathway in A. niger as compensatory response. By activation of this signaling pathway, cell wall reinforcing genes such as the agsA gene (encoding α-1,3-glucan synthase) become transcriptionally activated through the RlmA transcription factor, the ortholog of the S. cerevisiae Rlm1p protein ,,. Most surprisingly, this cell wall salvage mechanism is sufficient to ensure survival of A. niger when subjected to CFW  but not when stressed with the antifungal protein AFP. Although both compounds target cell wall chitin and induce expression of RlmA and its effector genes via the CWI pathway, this defense strategy is not the most appropriate one to protect A. niger against AFP. Instead, triggering the calcium/calcineurin signaling pathway which in turn induces expression of the chitin synthase gene chsD confers a higher protection to A. niger against AFP . These observations suggest that the CWI pathway of A. niger is, as in S. cerevisiae, not the only compensatory mechanism important for the repair of compromised cell walls.
To obtain a more comprehensive view on the cellular responses that allow A. niger to adapt to and survive to cell wall stress conditions, we characterized in this study its transcriptional adaptation program when stressed with the calcium/calcineurin signaling inhibitor FK506 and with the inhibitor of sphingolipid synthesis aureobasidin A (AbaA). Block of sphingolipid synthesis by AbaA has been shown to trigger protein kinase C signaling resulting in activation of Slt2p, the terminal MAP kinase of the CWI pathway in S. cerevisiae . The experimental approach of the present study was similar to that of our previous work, where we determined the transcriptomic fingerprint of A. niger when stressed with CA and FP, respectively . The identical approach allowed us to directly compare the data from both studies and enabled the identification of general survival and antifungal-specific stress responses. In brief, young germlings of A. niger were cultivated in bioreactors to ensure controlled, reproducible and equal growth conditions. Antifungals were added at sublethal concentrations that permitted A. niger to adapt to the growth-inhibitory effects and to respond with the formation of new (sub)apical branches. We also determined the transcriptomic and phenotypic consequences of inactivating the rlmA gene in A. niger. Overall, our transcriptomic data led to the conclusion that in addition to RlmA, MsnA, a predicted Msn2p orthologue, as well as CrzA are important for A. niger to withstand cell wall stress conditions. We therefore characterized the function of MsnA and CrzA for cell survival of A. niger by analyzing respective null strains.
The effect of AbaA (2 μg/ml) and FK506 (1,28 μg/ml) on A. niger germ tube formation and elongation
Mean BI (%)
Polarity axes (%)
Mean length (μM)
N = 1
N = 2
N = 3
3 ± 1
20.9 ± 0.9
13 ± 1
14.4 ± 0.1
5 ± 1
18.8 ± 1.8
11 ± 3
11.7 ± 0
FunCat classification of A. niger genes responsive to the treatment with caspofungin (CA), aureobasidin A (AbaA), FK506 and fenpropimorph (FP)
Sum of differentially expressed genes
Number of up-regulated genes
Number of down-regulated genes
Cell cycle and DNA processing
Protein with binding function or cofactor requirement
Cellular transport, transport facilitation and transport routes
Cellular communication/signal transduction mechanism
Cell rescue, defense and virulence
Interaction with the environment
Biogenesis of cellular components
Calcium has a regulatory function for several transporting steps in the constitutive secretory pathway in eukaryotes . Congruently, twelve genes predicted to function in protein folding, protein maturation and vesicle trafficking were differentially expressed upon FK506 treatment of A. niger germlings, eleven of which displayed increased expression and nine of which are also responsive genes to the treatment of A. niger with the ER-stressing agents DTT and tunicamycin , e.g. genes involved in signal peptide cleavage (An09g05420), protein translocation from and to the ER (An03g04940, An02g01510, An08g00740), glycosylation and quality control (An15g03330, An03g04410, An02g14930) or functioning as chaperones (An01g08420/ClxA and An01g04600/PrpA). Surprisingly, none of the FK506-responsive genes could be directly attributed to cell wall remodeling although two predicted G-protein coupled receptors were differentially expressed (An04g02930 and An02g01560/GprD). GprD shows similarity to the human LPA2 (EDG4) protein which acts as specific receptor for lysophosphatidic acid to activate calcium signaling and downstream protein kinase C . Supportively, GprD has been predicted to integrate stress signals via the calcineurin pathway in A. fumigatus  which, however has not been observed in A. nidulans .
Previously not identified was a group of four genes coding for proteins having a function in proteasomal degradation, i.e. An18g06700 (Pre7p ortholog), An18g06680 (Pre4p ortholog) and An04g01870 (Pre1p ortholog) and An14g00180 (Rpt6p ortholog; Additional file 5).
Transcriptome response of A. niger to fenpropimorph. FP is an inhibitor of ergosterol biosynthesis in S. cerevisiae by inhibiting sterol C-14 reductase (ERG24 gene) and sterol C-8 isomerase (ERG2 gene) . Expression of only 24 genes was modulated in A. niger upon FP treatment, among which was the predicted Erg2p ortholog, which suggests that FP exerts a similar mode of action in A. niger. (Figure 3B and C and Additional file 6). 14 of the responsive genes are indeed predicted to function in lipid metabolism and were all up-regulated upon FP stress: (i) SrbA, a transcription factor shown to control iron and ergosterol homeostasis in A. fumigatus , (ii) genes involved in β-oxidation (An08g05400, An17g01150, An15g01280, An08g07520, An16g04520, An14g00990), (iii) genes involved in fatty acid biosynthesis (An16g05340, An07g03290, An15g02830) and (iv) genes predicted to function in lipid transport across the peroxisomal, mitochondrial and plasma membrane (An18g01590, An04g00740, An01g12380). Hence, remodeling plasma membranes via lipid degradation and de novo synthesis might be the most appropriate compensatory response of A. niger to withstand FP-mediated inhibition of ergosterol homeostasis. In agreement, pyruvate carboxylase (An15g02820), a protein fueling the Krebs cycle was up-regulated, possibly reflecting the higher need of acetyl-CoA for fatty acid biosynthesis.
This dataset and its comparison with that of the wild-type led us to two hypotheses: First, RlmA is not the only cell wall stress related transcription factor as deletion of RlmA causes only down-regulation of one cell wall gene (agsA), whereas CA-induced stress in the wild-type induced expression of 21 cell wall genes. Second, inactivation of RlmA could have provoked a compensatory response in A. niger which confers a strong cross protection against cell wall stressors. In favor of the latter hypothesis are three observations: (i) Genes involved in cell wall remodeling, protein secretion and actin polarization were up-regulated in the deletion strain (Figure 5A and Additional file 7). (ii) The ΔrlmA strain did not show any hypersensitivity against CA, FP, AFP, FK506 and AbaA (Figure 4). (iii) Cultivation of the deletion strain in bioreactor settings and treatment with 50 ng/ ml CA did not provoke any significant transcriptomic response (data not shown), whereas it did for the wild-type strain (see above). It might be conceivable, however, that higher concentrations of CA would provoke a transcriptomic response in the ΔrlmA strain.
Promoter analysis of differentially expressed genes. In order to identify transcription factors, which in addition to RlmA mediate the survival response of A. niger to the cell surface stressors CA, FP, AbaA and FK506, we screened the 1,000-bp upstream regions of all differentially expressed genes for the presence of binding sites established for 25 transcription factors from different Aspergillus and Trichoderma species  and determined whether these motifs were significantly over- or underrepresented (500,000 bootstrap samples, FDR < 0.05). Binding sites for the transcription factors RlmA and MsnA were significantly enriched in the CA-responsive gene set of the wild-type strain, implying that MsnA could play in addition to RlmA an important role for the resistance of A. niger against cell wall stress while no binding sites were significantly enriched for either AbaA or FK506 treatment.
All three mutant strains became more sensitive towards CA, AbaA and CFW when cultivated on solid media (Figure 6), demonstrating that MsnA has - beside RlmA - a function in cell wall protection for A. niger. Whereas the contribution of both transcription factors was only very subtle with respect to 0.5 μg/ml CA and 2 μg/ml AbaA (note the ΔrlmA strain is insensitive to CA and AbaA when cultivated in liquid medium), both protect A. niger substantially against the chitin inhibitor CFW (40 μg/ml). Clearly, RlmA is the main contributor in the latter case because its inactivation resulted in a stronger growth-inhibited phenotype than inactivation of MsnA did. However, both seem to function in an additive or even synergistic manner, as the double mutant strains displayed a lethal phenotype on CFW plates. Notably, such an additive or synergistic phenotype was not observed on CA or AbaA plates, suggesting that both RlmA and MsnA function in the same signaling pathway when β-1,3-glucan polymerization or sphingolipid biosynthesis became inhibited.
Reinforcement of the cell wall is an essential survival response to shield cells after exposure to distinct cell surface stressors. Fungi have therefore developed various signaling pathways which sense and transmit the stress signal to the cell interior and the nucleus which in turn modulates gene expression such that the cell responds most appropriately to the life threatening condition. The well-studied unicellular yeast S. cerevisiae has been used as the main model system to study the underlying mechanisms. It has evolved at least three signaling pathways - the CWI pathway with its central components Pkcp, Slt2p and Rlm1p, the general response pathway with its mediators cAMP, PKA and Msn2/4 and the calcium/calcineurin pathway with its main effectors calcineurin and Crz1p – to reinforce its cell wall by increasing chitin, glucan and cell wall protein levels. These pathways are interwoven to maintain cell wall integrity during growth-mediated cell wall expansion and to flexible react to osmotic and mechanic stress conditions .
The main modules of cell wall salvage pathways are genetically fixed in yeast and filamentous fungi such as Aspergilli. However, accumulating evidences suggest that the individual modules differ in their cellular assignment, although the architectural hierarchy and direction of signal transmission is similar. To name just a few examples: The MsnA transcription factor is crucial for the stress response in S. cerevisiae but C. albicans , sensors of the CWI pathway process differently stress signals in A. fumigatus, A. nidulans, S. cerevisiae and Klyuveromyces lactis ,, the transcription factor CrzA does not act as activator of VCX1 expression in S. cerevisiae but of vcxA expression in A. nidulans , the Rho GTPases RacA and CdcA/Cdc42 differ fundamentally in their function in A. niger and A. nidulans  and the exocyst-mediated vesicle transport of S. cerevisiae and A. niger is only partially conserved . Hence, signal perception, transmission and translation can obviously differ among fungi which in fact could form a mechanistic explanation why fungi differ in their susceptibilities towards antifungal drugs. We have recently shown that the survival response of yeast and filamentous fungi towards the chitin synthase inhibitor AFP differs considerably. Whereas the presence of AFP provokes increased glucan synthesis via induction of the CWI pathway in AFP-sensitive fungi, AFP-resistant strains respond to AFP with enforced chitin synthesis by employing the calcium/calcineurin pathway . Hence, the outcome of an antifungal attack strongly depends on the species-specific survival strategy chosen, which causatively might be linked to the different use of signaling pathways and their modules.
The present work uncovered different defence strategies of A. niger to protect itself against cell wall stress conditions. At least three transcription factors - RlmA, MsnA and CrzA – are employed in an obvious sophisticated and well-balanced manner. The data also predicts a fourth factor, SrbA, which seems to be specifically important during cell membrane stress. Future studies will disclose how these regulators are interlocked in different signaling pathways to secure survival under different stress conditions.
Strains, growth conditions and antifungal compounds
Aspergillus niger strains used in this work
cspA1, amdS −
kusA::DR-amdS- DR, pyrG −
ΔkusA, pyrG + (derivative of MA70.15 containing A. niger pyrG)
ΔkusA, pyrG + , ΔrlmA ( derivative of AB4.1)
pyrG + (derivative of N402 containing A. niger pyrG)
ΔcrzA, pyrG + (derivative of MA234.1)
kusA::DR-amdS- DR, ΔrlmA, hph
kusA::DR-amdS-DR, msnA − , hph
kusA::DR-amdS- DR, msnA − , ΔrlmA, hph
Screening for antifungal-induced morphological changes
5 × 105 conidia of an A. niger strain were inoculated in Petri dishes containing 5 ml of liquid MM supplemented with 0.003% yeast extract. Prior to inoculation, coverslips were placed onto the bottom of the Petri dishes. Spores were allowed to germinate for 5 h at 37°C until small germ tubes became visible in more than 90% of the spores. Compounds were added at various concentrations. The negative control was supplemented with the same volume of solvent (ethanol or DMSO). After further cultivation for 1 h at 37°C, germlings that were adherent to the coverslips were analyzed by microscopy (see below). From at least 100 germlings per sample, the morphology was characterized as being either unbranched (germlings with a single germ tube) or branched (germlings with apical and/or subapical branches). The Branching Index was defined as BI = (Σ branched germlings) × (Σ branched + unbranched germlings)−1.
Growth assays in microtiter plates
104 conidia of an A. niger strain were inoculated in each well of 96-well optical glass bottom microtiter plates (Nunc art) in 200 μl MM supplemented with 0.003% yeast extract and cultivated for 30 hours at 30°C. Different concentrations of antifungals were supplemented prior to inoculation, whereby the negative controls were supplemented with the same volume of solvent (H2O, ethanol or DMSO). The effect of each compound was tested for at least 3 different concentrations in triplicates and each experiment was performed at least twice. Biomass accumulation was measured at fixed intervals at OD590.
Growth-plate inhibition assays
Defined spore titers of A. niger strains were used to inoculate MM plates supplemented with different concentrations of stress agents and incubated for 1–3 days at 30, 37 and 42°C, respectively. All experiments were performed at least in duplicates.
Freshly harvested conidia (5 × 108) from strain N402 were used to inoculate 0.5 liters of FM. Cultivations were performed in BioFlo/CelliGen 115 bioreactors (New Brunswick Scientific) as described earlier . In brief, 250 rpm was used as agitation speed and aeration was performed via the headspace until the dissolved oxygen tension dropped to 40%. Thereafter, aeration was switched to sparger aeration. Temperature and pH were set to 30°C and pH 3, respectively, and controlled on-line using the program NBS Biocommand. After 5 h of cultivation, AbaA (dissolved in 5 ml ethanol) or FK506 (dissolved in 5 ml DMSO) was added. 5 ml of ethanol or 5 ml of DMSO were added in the control runs. After an additional hour of cultivation, 500 ml of the culture broth were quickly harvested via filtration, and mycelial samples were immediately frozen using liquid nitrogen. In addition, samples were taken for microscopic analysis (see below) and calculation of the BI value. Note that the ΔrlmA deletion strain was cultivated 6 h instead of 5 h, as described previously .
Pictures of A. niger germlings were captured using an Axioplan 2 (Zeiss) equipped with a DKC-5000 digital camera (Sony). Light (using DIC settings) images were obtained with a 40× objective. Images were processed using Adobe Photoshop 6.0 (Adobe Systems Inc.).
RNA extraction, expression profiling
Total RNA isolation, RNA quality control, labeling, Affymetrix chip hybridization, scanning and signal calculation were performed as described previously . Microarray analyses were performed for biological duplicates for each condition (controls, FK506-, and AbaA-treated samples). Expression data was analyzed using the open source programs R (http://www.r-project.org/) and Bioconductor (http://www.bioconductor.org/). Background correction, normalization and probe summarization was performed using the default setting of the robust multi-array analysis (RMA) package as recently described . Differential gene expression was evaluated by moderated t-statistics using the Limma package  with a threshold of the Benjamini and Hochberg False Discovery Rate (FDR) of 0.05 . Fold change of gene expression from different samples was calculated from normalized expression values. Geometric means of the expression values as well as fold change for all conditions and comparisons are summarized in Additional file 1 and Additional file 2 and have been deposited at the GEO repository (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE56471.
Responsive genes in the antifungal-treated samples were functionally classified into FunCat categories as described previously ,. In silico analysis of putative transcription factor binding sites localized in the 1,000-bp upstream regions the responsive genes was performed using the transcription factor binding site finder (TFBSF) tool . In brief, the upstream regions of the differentially expressed genes were searched for the presence of putative binding sites recognized by 25 known transcription factors from Aspergillus or Trichoderma species (see Additional file 9). To determine significant over- or underrepresentation of binding sites, the background distribution of the identified motifs in the genome of A. niger was determined via bootstrapping (500,000 bootstraps).
Inactivation of rlmA and msnA genes in A. niger
To inactivate the rlmA gene, a deletion approach was followed as described previously . Construct pΔRlmA  was used to delete the rlmA gene of A. niger in the ΔkusA background strain MA169.4 . The plasmid was linearized with BglI prior to transformation. Transformants were purified twice on MM plates lacking uridine to obtain homokaryotic mycelium (pyrG+). Successful deletion was verified via Southern analysis (Additional file 10). Strain MF3.2 was selected for further analyses.
To inactivate the msnA gene, a disruption approach was followed. A 659 bp long fragment of the msnA gene comprising part of its 5’ open reading frame was amplified using the primers MsnA_fw_hind and MsnA_rev_hind (Additional file 11) and cloned into the unique restriction site HindIII of pAN7.1 . Using the hph gene for hygromycin B resistance as a selective marker, the resulting vector pJH1.56 was co-transformed together with pAB4.1  into MA169.4. This co-transformation approach was necessary to change the pyrG− genetic background of MA169.4 into pyrG+. Transformants were purified twice on MM plates containing 100 μg/ ml hygromycin B and lacking uridine to obtain homokaryotic mycelium (hygB, pyrG+). Correct disruption of the msnA disruption cassette was verified via Southern analysis (Additional file 10). Strain JH1.1 was selected for further analyses.
To obtain a strain in which both rlmA and msnA genes were inactivated, strain MF3.2 (ΔrlmA) was transformed with the msnA disruption construct pJH1.56. Transformants were purified twice on MM containing 100 μg/ ml hygromycin B. Correct disruption of the msnA disruption cassette was verified via Southern analysis (Additional file 10). Strain MF4.10 was selected for further analyses.
Availability of supporting data
The data sets supporting the results of this article are available in the GEO repository, (http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE56471.
This project was partly funded by the Marie Curie Integration grant to VM (CIG 303684) and supported by the Kluyver Centre for Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research. We are grateful to Johannes Heindorf and Janine Gündel for their excellent technical assistance.
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