Fungal Cell Wall Biogenesis Research Articles

Fungal Pathogenesis-Related Cell Wall Biogenesis, with Emphasis on the Maize Anthracnose Fungus Colletotrichum graminicola.

The genus Colletotrichum harbors many plant pathogenic species, several of which cause significant yield losses in the field and post harvest. Typically, in order to infect their host plants, spores germinate, differentiate a pressurized infection cell, and display a hemibiotrophic lifestyle after plant invasion. Several factors required for virulence or pathogenicity have been identified in different Colletotrichum species, and adaptation of cell wall biogenesis to distinct stages of pathogenesis has been identified as a major pre-requisite for the establishment of a compatible parasitic fungus–plant interaction. Here, we highlight aspects of fungal cell wall biogenesis during plant infection, with emphasis on the maize leaf anthracnose and stalk rot fungus, Colletotrichum graminicola.

Transglycosidases and Fungal Cell Wall β-(1,3)-Glucan Branching

Fungal cells are endowed with a cell wall that plays a crucial role in the fungal life, by providing mechanical strength and protecting fungal cells from their environment. Chemically, this fungal cell wall consists of different polysaccharides, contributing to 80-95% of the cell wall dry mass. The core cell wall structure is made up of β-(1,6)-branched β-(1,3)- glucan linked to chitin, and is common to all fungal species. Branching leads to β-(1,3)-glucan ramificating, facilitating its cross-linking with chitin as well as other cell wall components resulting in the construction of a functional fungal cell wall. Recently, using Saccharomyces cerevsiae as the model, we showed that the dual activity associated with CAZy family GH72 transglycosidases (http://www.cazy.org/) belonging to the GAS-family, are capable of elongating as well as branching β-(1,3)-glucan, an essential phenomenon during cell wall biogenesis and remodeling. Not only GAS-proteins, but also GEL-family protein from Aspergillus fumigatus (a pathogenic fungus) showed β-(1,3)- glucan elongating-branching activity. Interestingly, this dual activity was shown by only those GH72 family members containing a Carbohydrate Binding Motif-43 (CBM), suggesting that branching activity is universal in the fungal kingdom. Disruption of β-(1,3)-glucan branching resulted either in a sick-phenotype or led to inviability, suggesting that β-(1,3)-glucan branching is an essential phenomenon during fungal cell wall biogenesis. In this commentary, future perspectives on our findings are presented.

Genetic Manipulation of the Pneumocandin Biosynthetic Pathway for Generation of Analogues and Evaluation of Their Antifungal Activity.

Pneumocandins are lipohexapeptides of the echinocandin family that potently interrupt fungal cell wall biogenesis by noncompetitive inhibition of 1,3-β-glucan synthase. The pneumocandin biosynthetic gene cluster was previously elucidated by whole genome sequencing. In addition to the core nonribosomal peptide synthetase and polyketide synthase (GLNRPS4 and GLPKS4), the pneumocandin biosynthetic cluster includes two P450-type hemeprotein monooxygenase genes (GLP450-1 and GLP450-2) and four nonheme mononuclear iron oxygenase genes (GLOXY1, GLOXY2, GLOXY3, and GLOXY4), which function to biosynthesize and create the unusual sequence of hydroxylated amino acids of the mature pneumocandin peptide. Insertional inactivation of three of these genes (GLP450-1, GLP450-2, and GLOXY1) generated 13 different pneumocandin analogues that lack one, two, three, or four hydroxyl groups on 4R,5R-dihydroxy-ornithine and 3S,4S-dihydroxy-homotyrosine of the parent hexapeptide. Among them, seven analogues are previously unreported genetically engineered pneumocandins whose structures were established by NMR experiments. These new pneumocandins afforded a unique opportunity for side-by-side exploration of the effects of hydroxylation on pneumocandin antifungal activity. All of these cyclic lipopeptides showed potent antifungal activities, and two new metabolites pneumocandins F (3) and G (4) were more potent in vitro against Candida species and Aspergillus fumigatus than the principal fermentation products, pneumocandins A0 and B0.

SUN Proteins Belong to a Novel Family of β-(1,3)-Glucan-modifying Enzymes Involved in Fungal Morphogenesis

SUN proteins are involved in yeast morphogenesis, but their function is unknown. SUN protein plays a role in the Aspergillus fumigatus morphogenesis. Biochemical properties of recombinant SUN proteins were elucidated. Both Candida albicans and Aspergillus fumigatus sun proteins show a β-(1,3)-glucanase activity. The mode of action of SUN proteins on β-(1,3)-glucan is unique, new, and original. In yeasts, the family of SUN proteins has been involved in cell wall biogenesis. Here, we report the characterization of SUN proteins in a filamentous fungus, Aspergillus fumigatus. The function of the two A. fumigatus SUN genes was investigated by combining reverse genetics and biochemistry. During conidial swelling and mycelial growth, the expression of AfSUN1 was strongly induced, whereas the expression of AfSUN2 was not detectable. Deletion of AfSUN1 negatively affected hyphal growth and conidiation. A closer examination of the morphological defects revealed swollen hyphae, leaky tips, intrahyphal growth, and double cell wall, suggesting that, like in yeast, AfSun1p is associated with cell wall biogenesis. In contrast to AfSUN1, deletion of AfSUN2 either in the parental strain or in the AfSUN1 single mutant strain did not affect colony and hyphal morphology. Biochemical characterization of the recombinant AfSun1p and Candida albicans Sun41p showed that both proteins had a unique hydrolysis pattern: acting on β-(1,3)-oligomers from dimer up to insoluble β-(1,3)-glucan. Referring to the CAZy database, it is clear that fungal SUN proteins represent a new family of glucan hydrolases (GH132) and play an important morphogenetic role in fungal cell wall biogenesis and septation.

Efg1 Controls Caspofungin-Induced Cell Aggregation of Candida albicans through the Adhesin Als1

Echinocandin drugs such as caspofungin (CASP), micafungin, and anidulafungin inhibit fungal cell wall biogenesis by blocking Fks1-mediated β-glucan deposition into the cell surface. Candins have become suitable drugs to treat life-threatening diseases caused by several fungal species, including Candida albicans, that are pathogenic for humans. Here, we present the discovery of a novel CASP-induced flocculation phenotype of C. albicans, which formed large cell aggregates in the presence of CASP. High concentrations of sugars such as mannose or glucose inhibit CASP-induced flocculation and improve survival of C. albicans cells exposed to CASP. Notably, exposure of C. albicans cells to CASP triggers Efg1-dependent expression of the adhesin ALS1 and induces invasive growth on agar plates. Indeed, cells lacking either Efg1 or Als1 show strongly diminished CASP-induced flocculation, and the absence of Efg1 leads to marked CASP hypersensitivity. On the other hand, CASP-induced invasive growth is enhanced in cells lacking Efg1. Hence, CASP stress drives an Efg1-dependent response, indicating that this multifunctional transcriptional regulator, which is otherwise involved in filamentation, white-to-opaque switching, and virulence, also modulates cell wall remodeling upon CASP challenge. Taken together, our data suggest that CASP-induced cell wall damage activates Efg1 in parallel with the known cell integrity stress signaling pathway to coordinate cell wall remodeling.

Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay

BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.

Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post‐transcriptional regulator Ccr4‐Pop2

The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.

Biology of fungal cell walls—from single proteins to regulatory networks

The scientific success of the 4th International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, which was held in Warsaw, Poland, from 30 August until 3 September 2009, prompted us to work on this special issue of Yeast. It is the second such issue devoted to fungal cell wall biogenesis. The first one was published in 2007, following the ‘Cell Wall’ meeting in Heidelberg, Germany. In this present issue, the major topics discussed at the 2009 meeting in Warsaw are reflected. As indicated by the numbering, three meetings of this kind have been held previously, starting with the first one in 2001 (Ascona, Switzerland), followed by the second in 2003 (Salamanca, Spain) and the third in 2006 (Heidelberg, Germany). As usual, the top-ranking scientists working on the biology of fungal cell walls — with a species range from the classical model yeast Saccharomyces cerevisiae, through Schizosaccharomyces pombe and Candida albicans to filamentous fungi such as Aspergillus fumigatus — came together for lively discussions. Existing collaborations were further manifested and new ones were initiated. What makes fungal cell walls so special that they warrant such a series of international meetings? In recent decades, fungi have grown to be of great value as production organisms for all kinds of organic compounds, led by the model yeast S. cerevisiae in the field of heterologous protein production of pharmaceutical importance (e.g. interferon, hirudin, somatostatin, etc.). Although some of these proteins can be engineered to be secreted, especially if Pichia stipitis is employed as a yeast host, with many expression systems the respective proteins are retained within the cells. Purification of the desired product then requires cell disruption, which could be facilitated, and the results economically more appealing, if we better understood the biology of fungal cell wall biogenesis. Moreover, yeast glucans can be directly employed to boost the human immune system, to filter fungal toxins or for the improvement of wine quality during the production process. Again, understanding the regulatory circuits underlying glucan synthesis in vivo may have an enormous economic impact. Other fungi are known for their deleterious effects on human health and in agriculture. Two prominent examples are C. albicans, which is an opportunistic human pathogen, and A. fumigatus, which causes major concerns in clinics, with the growing use of immune suppressors for organ transplants, and the ‘natural’ immune deficiency in HIV patients. Clearly, the fungal cell wall

Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Fungal cell wall biogenesis: building a dynamic interface with the environment.

Fungal cell wall biogenesis: building a dynamic interface with the environment.

The Second International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, Salamanca, Spain, 27 August?1 September 2003

The Second Fungal Cell Wall Meeting brought together about 115 scientists in the beautiful, historical city of Salamanca. It all had started as an initiative some years ago by Yoshifumi Jigami, who brought together the first embryonic group of fungal glycobiologists and cell wall specialists in Tsukuba, Japan. The need was felt to have more such meetings on a formal basis and thanks to the combined efforts of Markus Aebi and Widmar Tanner, the first official meeting on fungal cell walls took place in Ascona, Switzerland in 2001. This year a Spanish team led by Angel Duran and Cesar Nombela hosted the second meeting in Salamanca. Although a majority of the 115 selected participants are working with Saccharomyces and Candida cell walls, the number of attendants studying other fungi including various mycelial species has considerably increased, thereby eliminating artificial divisions created by history. Without pretending to be complete, I would like to present some highlights of this exciting and well-organized meeting. For anyone interested in more details of the topics discussed below, or other preferred topics, I refer to the website of this meeting, which presents the abstracts of 62 talks and 40 posters [1]. For a general review of the area, the reader may consult [2]. As one of the most eminent researchers in this field and presumably also the best and longest known, Enrico Cabib set the scene in his keynote lecture by analyzing the close relationship between cell wall construction and morphogenesis. For example, the septation machinery of yeast cells can be viewed …