A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and... Show moreA characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and classical complementation approach. First, we characterized a newly isolated color mutant, colA, which lacked pigmentation resulting in white or colorless conidia. Pigmentation of the colA mutant was restored by a gene (An12g03950) which encodes a putative 4′phosphopantetheinyl transferase protein (PptA). 4′Phosphopantetheinyl transferase activity is required for the activation of Polyketide Synthases (PKSs) and/or Non-Ribosomal Peptide Synthases (NRPSs). The loci whose mutation resulted in fawn, olive, and brown color phenotypes were identified by complementation. The fawn phenotype was complemented by a PKS protein (FwnA, An09g05730), the ovlA mutant by An14g05350 (OlvA) and the brnA mutant by An14g05370 (BrnA), the respective homologs of alb1/pksP, ayg1 and abr1 in A. fumigatus. Targeted disruption of the pptA, fwnA, olvA and brnA genes confirmed the complementation results. Disruption of the pptA gene abolished synthesis of all polyketides and non-ribosomal peptides, while the naphtho-γ-pyrone subclass of polyketides were specifically dependent on fwnA, and funalenone on fwnA, olvA and brnA. Thus, secondary metabolite profiling of the color mutants revealed a close relationship between polyketide synthesis and conidial pigmentation in A. niger. Show less
Fungi are a very successful species and are distributed worldwide. However, the presence of fungi is not always desired. Filamentous fungi can grow on living or dead organic material and even... Show moreFungi are a very successful species and are distributed worldwide. However, the presence of fungi is not always desired. Filamentous fungi can grow on living or dead organic material and even inside the host. Current methods to prevent fungal growth are insufficient, causing fatality after fungal infections or loss of crops. The cell wall of a fungus is an intriguing component. It protects the cell from the harsh environment and determines the shape of the cell. Hence the cell wall is an essential component to the cell and provides an attractive target for antifungals. Additionally, the cell wall contains components only found in fungi, and the target is a desirable target as it is exposed on the outside of the cell. Currently, little is known about the cell wall of filamentous fungi. In order to design new or improved antifungal compounds, a better understanding of the fungal cell wall and of its adaptation to various conditions is required. In this thesis, we have used Aspergillus niger as a model filamentous fungus to study the biosynthesis of the fungal cell wall. The cell wall is a highly dynamic structure and able to adapt to various changes, either developmental (e.g. mating, growth, budding, branching and sporulation), environmental (e.g. heat, pH, osmolarity, chemical compounds), or genetic (e.g. mutations in cell-wall related genes). In chapter 1 an overview is presented of the current state of knowledge about the fungal cell wall. The architecture, biosynthesis and the remodeling are discussed in this chapter. The response of our model fungus A. niger to chemical induced cell wall stress is described in chapter 2. The fluorescent brightener Calcofluor White (CFW) was used to induce cell wall stress. We show that A. niger, like Saccharomyces cerevisiae, responds to cell wall stress by an increase of chitin deposition in the cell walls. This increase in chitin, a structural cell wall polymer, was accompanied by an increased transcription level of gfaA. It was also shown that this mechanism is not only limited to A. niger but is also observed in other filamentous fungi like the plant pathogenic fungus Fusarium oxysporum and the food spoilage fungus Penicillium chrysogenum. It is further shown that gfaA is an essential gene and the deletion strain can be rescued by addition of glucosamine. In chapter 3, a family of five 1,3-__-D-glucan synthase encoding genes is described. The expression of these genes during various types of cell wall stress was monitored and it was found that the expression of agsA and agsE was induced. The induction of an 1,3-__-D-glucan synthase encoding gene after cell wall stress was also observed in P. chrysogenum. The deletion of agsA led to an increased sensitivity towards CFW. While in chapter 2 and 3 changes in expression levels of genes encoding proteins involved in cell wall biosynthesis are described, the mechanism behind the induction of cell wall stress responsive genes is described in chapter 4. A promoter deletion study combined with an in silico analysis indicated that the induction of agsA in response to cell wall stress is dependent on a putative Rlm1p binding site in its promoter. Therefore a gene, named rlmA encoding for a MADS-box transcription factor was isolated from A. niger after database searches. The role of this gene in the induction of agsA and gfaA after CFW stress was investigated. A deletion of the rlmA gene was constructed and this resulted in an increased sensitivity towards cell wall disturbing compounds. In S. cerevisiae an important part of the response towards cell wall threatening conditions is the up-regulation of GPI-anchored cell wall proteins. In chapter 5 the isolation and characterisation of an HF-extractable cell wall protein from A. niger, named CwpA, is described. It was shown by simple fractionation experiments that the protein was mainly present in the cell wall fraction. Deletion of cwpA resulted in an increased sensitivity towards CFW suggesting a structural role for CwpA. Chapter 6 describes a novel method for the identification of cell wall mutants. The mutants are first selected based on their compensatory reaction (induction of agsA) and subsequenly subjected to various secondary screens, to confirm an altered cell wall integrity. Four out of 240 mutants with induced agsA expression levels, named miaA-D, were selected for complementation. All four mutants were complemented by cosmids. Further subcloning experiments are underway to identify the mutated genes. In chapter 7 a GFP-based reporter system is described. The system allows the rapid screening of compounds to see if they trigger the cell wall integrity pathway and thereby induce the PagsA(-H2B)-GFP reporter. The method has been evaluated towards various putative antifungal compounds and is a promising tool for the identification of new cell wall related antifungal compounds. In conclusion, this thesis provides evidence for the existence of a cell wall remodeling mechanism in filamentous fungi and in particular A. niger. Also, signal transduction pathway components were identified by which cell wall weakening is sensed and transduced into a transcriptional response. Additionally, a cell wall stress reporter system was developed to identify new cell wall related antifungal targets and to identify cell wall related antifungal compounds. Show less
Damveld, R.A.; Arentshorst, M.; Kuyk, P.A. van; Klis, F.M.; Hondel, C.A.M.J.J. van den; Ram, A.F.J. 2005
Glycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell... Show moreGlycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell wall (GPI-CWPs). GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bond that is present in the remnant of the GPI-anchor. The filamentous fungus Aspergillus niger contains at least seven HF-extractable cell wall mannoproteins. One gene encoding an HF-extractable cell wall mannoprotein, cwpA, was cloned and further characterised. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences both at the N-terminus and C-terminus of the protein, for entering the ER and the addition of a GPI-anchor, respectively. A CwpA-specific antiserum was raised and in combination with fractionation experiments, we show that this protein was abundantly present as an HF-extractable protein in the cell wall of A. niger. Show less
Damveld, R.A.; Kuyk, P.A. van; Arentshorst, M.; Klis, F.M.; Hondel, C.A.M.J.J. van den; Ram, A.F.J. 2005
