Tomonori Fujioka

MpkA-Dependent and -Independent Cell Wall Integrity Signaling in Aspergillus nidulans

ABSTRACT Cell wall integrity signaling (CWIS) maintains cell wall biogenesis in fungi, but only a few transcription factors (TFs) and target genes downstream of the CWIS cascade in filamentous fungi are known. Because a mitogen-activated protein kinase (MpkA) is a key CWIS enzyme, the transcriptional regulation of mpkA and of cell wall-related genes (CWGs) is important in cell wall biogenesis. We cloned Aspergillus nidulans mpkA; rlmA, a TF gene orthologous to Saccharomyces cerevisiae RLM1 that encodes Rlm1p, a major Mpk1p-dependent TF that regulates the transcription of MPK1 besides that of CWGs; and Answi4 and Answi6, homologous to S. cerevisiae SWI4 and SWI6, encoding the Mpk1p-activating TF complex Swi4p-Swi6p, which regulates CWG transcription in a cell cycle-dependent manner. A. nidulans rlmA and mpkA cDNA functionally complemented S. cerevisiae rlm1Δ and mpk1Δ mutants, respectively, but Answi4 and Answi6 cDNA did not complement swi4Δ and swi6Δ mutants. We constructed A. nidulans rlmA, Answi4 and Answi6, and mpkA disruptants (rlmAΔ, Answi4Δ Answi6Δ, and mpkAΔ strains) and analyzed mpkA and CWG transcripts after treatment with a β-1,3-glucan synthase inhibitor (micafungin) that could activate MpkA via CWIS. Levels of mpkA transcripts in the mutants as well as those in the wild type were changed after micafungin treatment. The β-glucuronidase reporter gene controlled by the mpkA promoter was expressed in the wild type but not in the mpkAΔ strain. Thus, mpkA transcription seems to be autoregulated by CWIS via MpkA but not by RlmA or AnSwi4-AnSwi6. The transcription of most CWGs except α-1,3-glucan synthase genes (agsA and agsB) was independent of RlmA and AnSwi4-AnSwi6 and seemed to be regulated by non-MpkA signaling. The transcriptional regulation of mpkA and of CWGs via CWIS in A. nidulans differs significantly from that in S. cerevisiae.

Disordered Cell Integrity Signaling Caused by Disruption of the kexB Gene in Aspergillus oryzae

ABSTRACT We isolated the kexB gene, which encodes a subtilisin-like processing enzyme, from a filamentous fungus, Aspergillus oryzae. To examine the physiological role of kexB in A. oryzae, we constructed a kexB disruptant (ΔkexB), which formed shrunken colonies with poor generation of conidia on Czapek-Dox (CD) agar plates and hyperbranched mycelia in CD liquid medium. The phenotypes of the ΔkexB strain were restored under high osmolarity in both solid and liquid culture conditions. We found that transcription of the mpkA gene, which encodes a putative mitogen-activated protein kinase involved in cell integrity signaling, was significantly higher in ΔkexB cells than in wild-type cells. The ΔkexB cells also contained higher levels of transcripts for cell wall-related genes encoding β-1,3-glucanosyltransferase and chitin synthases, which is presumably attributable to cell integrity signaling through the increased gene expression of mpkA. As expected, constitutively increased levels of phosphorylated MpkA were observed in ΔkexB cells on the CD plate culture. High osmotic stress greatly downregulated the increased levels of both transcripts of mpkA and the phosphorylated form of MpkA in ΔkexB cells, concomitantly suppressing the morphological defects. These results suggest that the upregulation of transcription levels of mpkA and cell wall biogenesis genes in the ΔkexB strain is autoregulated by phosphorylated MpkA as the active form through cell integrity signaling. We think that KexB is required for precise proteolytic processing of sensor proteins in the cell integrity pathway or of cell wall-related enzymes under transcriptional control by the pathway and that the KexB defect thus induces disordered cell integrity signaling.

Functional analysis of C2H2 zinc finger transcription factor CrzA involved in calcium signaling in Aspergillus nidulans.

Calcium signaling systems are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. Calcineurin is an important signaling component, which mediates ion homeostasis and virulence in several fungi. Based on intensive studies conducted on budding yeast, transcription factor Crz1p is thought to be a target of calcineurin. To provide insight into calcium signaling, a Crz1p homolog (CrzA) in a filamentous fungus Aspergillus nidulans was identified and its function with special reference to calcium response was characterized. A crzA gene disruption mutant exhibited sensitivity to high concentrations of Mn2+ and Ca2+, and mediated the expression of P-type calcium-ATPase homologous genes. Comprehensive transcriptional analysis with DNA microarrays indicated that CrzA regulates the expression of a vacuolar Ca2+/H+ exchanger gene in response to external calcium stimuli. It is suggested that the calcineurin-CrzA pathway is the mediator of Ca2+ homeostasis in A. nidulans. Moreover, a crzA/hogA double mutant showed hypersensitivity to osmotic stress, indicating the importance of calcium homeostasis for adaptation to osmotic stress, a universal stress in filamentous fungi.

Functional analysis of the α-1,3-glucan synthase genes agsA and agsB in Aspergillus nidulans: agsB is the major α-1,3-glucan synthase in this fungus.

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and 13C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.

Novel Reporter Gene Expression Systems for Monitoring Activation of the Aspergillus nidulans HOG Pathway

The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a β-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogAΔ. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogAΔ mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.

Mitogen-activated protein kinases MpkA and MpkB independently affect micafungin sensitivity in Aspergillus nidulans

The transcriptional regulation of the MAPK mpkA and cell wall-related genes in Aspergillus nidulans differs from that of their counterparts in Saccharomyces cerevisiae. The A. nidulans MAPK MpkB is putatively orthologous to the yeast MAPKs Kss1p and Fus3p. To investigate MpkB and its contribution to cell wall integrity in A. nidulans, we constructed mpkB-disruptant (mpkB∆) strains. We previously showed that mpkA∆ strains exhibited reduced colony growth and increased sensitivity to the β-1,3-glucan synthase inhibitor micafungin. Like mpkA∆ strains, mpkB∆ strains exhibited slight growth retardation and increased sensitivity to micafungin. Although MpkB-dependent signaling modulated the transcription of some cell wall-related genes, the sugar composition of cell wall fractions was similar among wild-type, mpkA∆, and mpkB∆ strains. To elucidate the relationship between MpkA and MpkB pathways, we compared conditional mutants of mpkB with those with mpkA deletion. Sensitivity testing suggested that MpkA and MpkB additively contribute to micafungin activity in A. nidulans. Graphical Abstract Phenotypic analyses of the A. nidulans mpkA∆, mpkB∆, and conditional-mpkB with mpkA∆ strains revealed that the MpkA and MpkB additively contribute to micafungin activity. (24 words, 147 characters)

Alternative Processing of Proproteins in Aspergilli kexB Gene Disruptants under Hyperosmotic Conditions

Disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae and Aspergillus nidulans led to remarkable morphological defects, and these phenotypes were suppressed under hyperosmotic conditions. In this study, we investigated to determine whether non-KexB proteases might complement the in vivo function of KexB in the two Aspergillus kexB disruptants. Neither overexpression of opsA or opsB encoding A. oryzae aspartyl proteases homologous to yeast yapsins (YPS1/2) suppressed the kexB mutation, although yapsins are multicopy suppressors for the yeast kex2 mutation. A. nidulans and A. oryzae kexB disruptants grown under hyperosmotic conditions processed a recombinant fusion protein carrying a synthetic dibasic processing site (Lys-Arg) although the disruptants grown under normal growth conditions did not cleave the site. These results suggest that the two Aspergilli have other potential processing proteases that are induced and/or activated under hyperosmotic conditions and consequently complement, at least in part, the in vivo function of KexB.

Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae

A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.