Vast efforts have been devoted to the development of antifungal drugs targeting the cell wall, but the supramolecular architecture of this carbohydrate-rich composite remains insufficiently understood. Here we compare the cell wall structure of a fungal pathogen Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. High-resolution solid-state NMR spectroscopy of intact cells reveals a rigid core formed by chitin, β-1,3-glucan, and α-1,3-glucan, with galactosaminogalactan and galactomannan present in the mobile phase. Gene deletion reshuffles the composition and spatial organization of polysaccharides, with significant changes in their dynamics and water accessibility. The distribution of α-1,3-glucan in chemically isolated and dynamically distinct domains supports its functional diversity. Identification of valines in the alkali-insoluble carbohydrate core suggests a putative function in stabilizing macromolecular complexes. We propose a revised model of cell wall architecture which will improve our understanding of the structural response of fungal pathogens to stresses.

No abstract

Chitinases are produced throughout the growth process of fungi and are thought to play important roles in morphogenesis. Aspergillus fumigatus, is an important pathogen of immunocompromised individuals in which it causes pneumonia and invasive disseminated disease with high mortality; it is also known to produce chitinase. We have induced an exceptionally stable extracellular chitinase in A. fumigatus YJ-407, which could be isolated readily in a homogeneous form by using ammonium sulfate precipitation followed by DEAE-cellulose chromatography and preparative PAGE. The molecular mass of this chitinase was estimated to be 46 000 by SDS/PAGE, and its isoelectric point was pH 5.6. The enzyme was most active at pH 5.0 and 60 8C, and was inhibited strongly by Hg . The enzyme was stable over a broad pH range 4±8 and below 45 8C. Tryptophan and carboxyl groups were found to be essential for the enzyme activity. The Michaelis constants for swollen chitin and chitosan were 1.12 mg´mL 21 and 1.84 mg´mL 21 , respectively. The enzyme showed maximum activity towards glycol chitin and partially deacetylated chitosan, and lower activity towards colloidal chitin. Analysis of the hydrolysis product showed that the enzyme has both endoand exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.Keywords: Aspergillus fumigatus; chitinase; endochitinase; exochitinase; transglycosylation.Chitinases, enzymes that cleave the bond between the C1 and C4 of two consecutive N-acetylglucosamines of chitin, have been found in microorganisms, plants and animal tissues. These enzymes have been shown to have a variety of functions. In plants, which lack chitin, chitinases are thought to be a defence system against fungal pathogens. Chitinases produced by bacteria appear to have a nutritional or scavenging role, while those produced by filamentous fungi have been shown to be involved in a variety of functions such as cell wall digestion, germination of spores, hyphal growth, hyphal autolysis, differentiation into spores, assimilation of chitin and mycoparasitism [1±3]. However, their roles in fungal growth and mechanisms of chitinase regulation are almost totally unknown.As one of the most ubiquitous of the airborne saprophytic fungi, Aspergillus fumigatus has been shown to be an opportunistic pathogen causing pneumonia and other fatal invasive infections in immunocompromised hosts, particularly among patients undergoing cytotoxin chemotherapy or bone-marrow transplantation [4±6]. There has been a dramatic increase in severe and usually fatal invasive aspergillosis caused by A. fumigatus. This organism is also known to secrete extracellular chitinases [7,8]. To understand the pathogenesis associated with A. fumigatus infection, we have studied the chitinases in this organism. An extracellular chitinase, which was remarkably resistant to physical and chemical manipulations, was induced from A. fumigatus YJ-407 and could be isolated readily in homogeneous form by ammonium sulfate precipitation followed by DEAE-cellulose c...