Expression of agsA, one of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress

1,3-alpha-D-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-alpha-D-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-alpha-D-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi.     

A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative alpha-glucan synthase, is strongly induced in response to cell wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that cell wall mutants with a constitutively active cell wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various cell wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for fungal cell wall biosynthesis and represents an attractive target for the development of antifungals.     

Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red

The fungal cell wall is an essential organelle and represents a considerable metabolic investment. Its macromolecular composition, molecular organization and thickness can vary greatly depending on environmental conditions. Its construction is also tightly controlled in space and time. Many genes are therefore involved in building the fungal cell wall. Here we present a simple approach for detecting these genes. The method is based on the observation that cell wall mutants are generally more sensitive to two related anionic dyes, Calcofluor white (CFW) and Congo red (CR), both of which interfere with the construction and stress response of the cell wall. CFW-based and CR-based susceptibility assays identify cell wall mutants not only in ascomycetous yeasts (such as Saccharomyces cerevisiae and Candida albicans) but also in mycelial ascomycetes (such as Aspergillus fumigatus and Aspergillus niger), basidiomycetous species (Cryptococcus neoformans) and probably also zygomycetous fungi. The protocol can be completed in 4-6 h (excluding the incubation time required for fungal growth).     

Vacuolar H(+)-ATPase plays a key role in cell wall biosynthesis of Aspergillus niger

The identification of suitable targets is crucial for the discovery and development of new antifungals. Since the fungal cell wall is an essential organelle, the identification of genes involved in cell wall biosynthesis is expected to help discover new antifungal targets. From our previously obtained collection of cell wall mutants with a constitutively active cell wall stress response pathway, we selected a thermosensitive, osmotic-remediable mutant with decreased resistance to SDS for complementation analysis. The phenotypes of this mutant were complemented by a gene encoding a protein with high sequence similarity to subunit d of the eukaryotic Vacuolar-H(+)-ATPase (VmaD). Genetic analysis of this thermosensitive mutant revealed that the conditional mutant allele encodes a protein that lacks 12 amino acids at the C-terminus due to a point mutation that introduces a stop codon. Deletion of the entire gene resulted in very poor growth. The conditional mutant displayed several phenotypes that are typical for V-ATPase mutants, including increased sensitivity to zinc ions and reduced acidification of the vacuole as observed by quinacrine staining. Treatment of Aspergillus niger with the V-ATPase inhibitor bafilomycinB(1) induced the expression of agsA and other cell wall related genes. Furthermore genes involved in cell wall reassembly like fksA, agsA and phiA were clearly up-regulated in the conditional mutant. Our results indicate that the ATP-driven transport of protons and acidification of the vacuole is crucial for the strength of the fungal cell wall and that reduced activity of the V-ATPase induces the cell wall stress response pathway.     

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.