Trypacidin, a spore-borne toxin from Aspergillus fumigatus, is cytotoxic to lung cells

Inhalation of Aspergillus fumigatus conidia can cause severe aspergillosis in immunosuppressed people. A. fumigatus produces a large number of secondary metabolites, some of which are airborne by conidia and whose toxicity to the respiratory tract has not been investigated. We found that spores of A. fumigatus contain five main compounds, tryptoquivaline F, fumiquinazoline C, questin, monomethylsulochrin and trypacidin. Fractionation of culture extracts using RP-HPLC and LC-MS showed that samples containing questin, monomethylsulochrin and trypacidin were toxic to the human A549 lung cell line. These compounds were purified and their structure verified using NMR in order to compare their toxicity against A549 cells. Trypacidin was the most toxic, decreasing cell viability and triggering cell lysis, both effects occurring at an IC₅₀ close to 7 µM. Trypacidin toxicity was also observed in the same concentration range on human bronchial epithelial cells. In the first hour of exposure, trypacidin initiates the intracellular formation of nitric oxide (NO) and hydrogen peroxide (H₂O₂). This oxidative stress triggers necrotic cell death in the following 24 h. The apoptosis pathway, moreover, was not involved in the cell death process as trypacidin did not induce apoptotic bodies or a decrease in mitochondrial membrane potential. This is the first time that the toxicity of trypacidin to lung cells has been reported.     

Biochemical characterisation of ketoconazole inhibitory action on Aspergillus fumigatus

Abstract The effect of ketoconazole on growth, sterol composition, in vitro sterol biosynthesis and P450-CO complex formation and its interaction with microsomal P450 was determined. On solid medium and in liquid medium ketoconazole inhibited Aspergillus fumigatus growth completely at 5 × 10⁻⁵ M and 50% of the growth at 1.3 × 10⁻⁵ M and 2.1 × 10⁻⁵ M respectively. A close relationship between accumulation of 14α-methyl sterols (eburicol, obtusifoliol and 14α-methyl fecosterol) and depletion of ergosterol with growth arrest was observed in ketoconazole treated cultures. The half inhibitory concentration for in vitro ergosterol biosynthesis and half saturating concentration for type II binding spectrum of ketoconazole were calculated as 73.8 ± 6.3 nM and 0.13 ± 0.04 μM respectively. CO displacement studies revealed inhibition of CO-P450 complex formation by ketoconazole.     

Galactosaminogalactan secreted from Aspergillus fumigatus and Aspergillus flavus induces platelet activation

Platelets are meanwhile recognized as versatile elements within the immune system and appear to play a key role in the innate immune response to pathogens including fungi. Previous experiments revealed platelet activation by direct contact with the hyphal-associated polysaccharide galactosaminogalactan (GAG). Since secreted fungal products may also be relevant and trigger immune reactions or thrombosis, we screened culture supernatants (SN) of human-pathogenic fungi for their capacity to activate platelets. For that purpose, platelets were incubated with SN from various fungal species; platelet activation and GAG deposition on the surface of platelets were detected by flow cytometry and electron and confocal microscopy, Culture supernatants of Aspergillus fumigatus and flavus isolates were potent platelet stimulators in a dose- and time-dependent manner, while SN of other Aspergillus species and all tested mucormycete species did not significantly induce platelet activation. The capacity of culture SN to activate platelets was dependent on fungal production of GAG and deposition of secreted GAG on the platelet surface; supernatants from mucormycetes or mutants of A. fumigatus lacking GAG secretion did not affect platelet activity. These results suggest that invading fungi can stimulate platelets not only locally through direct interactions with fungal hyphae, but can also act over a certain distance through secreted GAG.     

A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407.

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 degrees C, and was inhibited strongly by Hg2+, Pb2+, Ag+, Fe2+, Mn2+ and Zn2+. The enzyme was stable over a broad pH range 4-8 and below 45 degrees C. 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-1 and 1.84 mg.mL-1, 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 endo- and exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.     

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.     

Isolation and toxigenicity of Aspergillus fumigatus from moldy silage

Thirty-nine silage samples were collected from various siloson Terceira Island in the Azores. Samples were examined for the presence of total fungi, and isolates of Aspergillus fumigatus were analyzed for their ability to produce fumitremorgens B and C, fumigaclavines B and C, and gliotoxin. Thirty-four silage samples (87%) were contaminated with fungi, and A. fumigatus was isolated from 27 samples (69%). Samples that were taken from the surface of silos had significantly higher populations of both total fungi and A. fumigatus than did samples taken from the middle of silos. Analysis of 27 A. fumigatus isolates (one representing each positive sample) showed that 59.3% produced fumitremorgen B; 33.3% produced fumitremorgen C; 29.6% produced fumigaclavine B; 7.4% produced fumigaclavine C; and 11.1% produced gliotoxin. Fifty-two percent of the isolates produced multiple toxins, and 25.9% did not produce any of these toxins. Gliotoxin and fumigaclavine C were always produced in combination with other toxins. Because of the demonstrated potential of these A. fumigatus isolates to producemycotoxins, it is important to properly construct and manage silos to prevent their contamination with A. fumigatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

Galactomannoproteins of Aspergillus fumigatus

Galactofuranose-containing molecules have been repeatedly shown to be important antigens among human fungal pathogens, including Aspergillus fumigatus. Immunogenic galactofuran determinants have been poorly characterized chemically, however. We reported here the characterization of two glycoproteins of A. fumigatus with an N-glycan containing galactofuranose. These proteins are a phospholipase C and a phytase. Chemical characterization of the N-glycan indicates that it is a mixture of Hex(5-13)HexNAc(2) oligosaccharides, the major molecular species corresponding to Hex(6-8)HexNAc(2). The N-glycan contained one galactofuranose unit that was in a terminal nonreducing position attached to the 2 position of Man. This single terminal nonreducing galactofuranose is essential for the immunoreactivity of the N-glycans assessed either with a monoclonal antibody that recognizes a tetra-beta-1,5-galactofuran chain of galactomannan or with Aspergillus-infected patient sera.     

Glycosylinositolphosphoceramides in Aspergillus fumigatus

Fungal glycosylinositolphosphoceramides (GIPCs) are involved in cell growth and fungal-host interactions. In this study, six GIPCs from the mycelium of the human pathogen Aspergillus fumigatus were purified and characterized using Q-TOF mass spectrometry and 1H, 13C, and 31P NMR. All structures have the same inositolphosphoceramide moiety with the presence of a C(18:0)-phytosphingosine conjugated to a 2-hydroxylated saturated fatty acid (2-hydroxy-lignoceric acid). The carbohydrate moiety defines two types of GIPC. The first, a mannosylated zwitterionic glycosphingolipid contains a glucosamine residue linked in alpha1-2 to an inositol ring that has been described in only two other fungal pathogens. The second type of GIPC presents an alpha-Manp-(1-->3)-alpha-Manp-(1-->2)-IPC common core. A galactofuranose residue is found in four GIPC structures, mainly at the terminal position via a beta1-2 linkage. Interestingly, this galactofuranose residue could be substituted by a choline-phosphate group, as observed only in the GIPC of Acremonium sp., a plant pathogen.     

烟曲霉与宿主细胞相互作用规律的初步研究

目的研究烟曲霉侵入宿主细胞过程中的基本规律及宿主细胞肌动蛋白骨架的变化情况。方法利用表达绿色荧光的烟曲霉ATCC13073,研究烟曲霉侵入上皮细胞和被巨噬细胞吞噬随时间的变化规律。结果烟曲霉侵入吞噬细胞和非吞噬细胞的量随时间呈现完全不同的规律,烟曲霉侵入上皮细胞A549能力较弱,在7h后侵染量才有明显的增加,为原始接种量的（0.09±0.01）%。前3h,鼠巨噬细胞J774吞噬量迅速升高,然后侵染量缓慢下降。烟曲霉侵入宿主细胞过程中会引起宿主细胞肌动蛋白骨架的重排,侵入过程中伴有吞噬杯的形成。结论巨噬细胞吞噬烟曲霉和烟曲霉侵入上皮细胞规律有明显不同,侵入过程都伴随肌动蛋白骨架的重排。