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.     

Invasive pulmonary aspergillosis due to a mixed infection caused by Aspergillus flavus and Aspergillus fumigatus

Invasive pulmonary aspergillosis is typically caused by a single Aspergillus species, most frequently Aspergillus fumigatus. Here we report that a lung transplant recipient developed invasive aspergillosis due to a mixed infection caused by Aspergillus flavus and A. fumigatus. The implications for this unusual finding are discussed.     

Biosynthesis of extracellular and intracellular gold nanoparticles by Aspergillus fumigatus and A. flavus

Green chemistry is a boon for the development of safe, stable and ecofriendly nanostructures using biological tools. The present study was carried out to explore the potential of selected fungal strains for biosynthesis of intra- and extracellular gold nanostructures. Out of the seven cultures, two fungal strains (SBS-3 and SBS-7) were selected on the basis of development of dark pink colour in cell free supernatant and fungal beads, respectively indicative of extra- and intracellular gold nanoparticles production. Both biomass associated and cell free gold nanoparticles were characterized using X-ray diffractogram (XRD) analysis and transmission electron microscopy (TEM). XRD analysis confirmed crystalline, face-centered cubic lattice of metallic gold nanoparticles along with average crystallite size. A marginal difference in average crystallite size of extracellular (17.76 nm) and intracellular (26 and 22 nm) Au-nanostructures was observed using Scherrer equation. In TEM, a variety of shapes (triangles, spherical, hexagonal) were observed in both extra- and intracellular nanoparticles. 18S rRNA gene sequence analysis by multiple sequence alignment (BLAST) indicated 99 % homology of SBS-3 to Aspergillus fumigatus with 99 % alignment coverage and 98 % homology of SBS-7 to Aspergillus flavus with 98 % alignment coverage respectively. Native-PAGE and activity staining further confirmed enzyme linked synthesis of gold nanoparticles.     

Chromatographic fractionation of Aspergillus endotoxins

Summary Crude preparations of the endotoxins extracted from the mycelia ofAspergillus fumigatus andAspergillus flavus, after preliminary concentration by ammonium sulfate, have been fractionated by column chromatography on DEAE-cellulose. Although the biological activity of the chromatographed preparations was not limited to a single fraction, examination of the most active fractions by starch gel electrophoresis showed no bands common to the two nephrotoxins.The highest hemolytic and toxic activities of the fumigatus toxin were found in different fractions of the chromatographed material, and starch gel electrophoresis showed no bands common to these two fractions.The molecular weight of the flavus toxin has been estimated to be in the range of 32,000 to 34,000 as judged by the results of ultracentrifugation and microelectrodialysis in starch gels of the most toxic fractions.Both of the toxins have been shown to contain small amounts of hexosamine and larger amounts of non-amino sugars.Presented in part at the 46th Annual Meeting of the American Society of Biological Chemists, Atlantic City, New Jersey, April, 1962.     

黄曲霉菌原生质体的制备和再生技术优化

黄曲霉菌的遗传转化是研究黄曲霉菌致病相关功能基因的前提和基础,而原生质体是研究和建立真菌遗传转化系统的重要工具。本文分别以黄曲霉孢子和菌丝为材料,研究不同条件下黄曲霉原生质体的形成和再生,结果表明,黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3 h,原生质体制备率高达97.3%,再生率达89.2%;黄曲霉菌丝在菌龄为42 h,酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解1 h,可获得最高原生质体产量为2.0×10^6个/m L,再生培养基中以1 mol/L蔗糖作为渗透压稳定剂时,原生质体再生率达5.5%。故本实验条件下,黄曲霉孢子原生质体的形成和再生优于菌丝。     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

Purification and properties of a heat-stable exoinulinase isoform from Aspergillus fumigatus

An inducible extracellular exoinulinase (isoform II) was purified from the extracellular extract of Aspergillus fumigatus by ammonium sulphate precipitation, followed by successive chromatographies on DEAE-Sephacel, Octyl-Sepharose (HIC), Sephacryl S-200, affinity chromatography on ConA-CL Agarose and Sephacryl S-100 columns. The enzyme was purified 75-folds with 3.2% activity yield from the starting culture broth. The purified isoform II was a monomeric 62 kDa protein with a pI value of 4.5. The enzyme showed maximum activity at pH 6.0 and was stable over a pH range of 4.0-7.0, whereas the optimum temperature for enzyme activity was 60 degrees C. The inulinase isoform II showed exo-inulinolytic activity and retained 72% and 44% residual activity after 12 h at 60 degrees C and 70 degrees C, respectively. The inulin hydrolysis activity was completely abolished with 5 mM Hg2+ and Fe2+, whereas K+ and Cu2+ enhanced the inulinase activity. As compared to sucrose, stachyose and raffinose the purified enzyme had a lower Km (1.25 mM) and higher catalytic center activity (Kcat = 3.47 x 10(4) min(-1)) for inulin. As compared to exoinulinase isoform I of A. fumigatus, purified earlier, the isoform II is more thermostable and is a potential candidate for commercial production of fructose from inulin.     

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.     

烟曲霉基因组与致病机制

烟曲霉(Aspergillus fumigatus)是环境中普遍存在的丝状腐生真菌,也是重要的机会致病菌。随着免疫受损人群的增多,由烟曲霉引起的系统性感染发病率不断升高,已成为重症免疫受损患者死亡的主要原因之一。文章结合烟曲霉全基因组序列信息对烟曲霉感染过程、致病机制等做一概述。     

Bacterial Antagonists of Aspergillus flavus

In order to search for bacteria capable of reducing the aflatoxin contamination of cottonseed, 892 indigenous bacterial isolates, including 11 that were endophytic to cotton, were screened for their ability to inhibit the growth of Aspergillus flavus on cottonseed in an in vitro bioassay. Only six isolates partially or totally inhibited fungal growth. All antagonistic isolates were recovered from boll, lint or seed surface or from the lint of mature bolls. One was retrieved from mature seeds. None of the endophytic isolates showed activity. In four field trials, the incidence of A. flavus -induced damage to locules inoculated simulteously with A. flavus plus the most A. flavus plus the most effective antagonistic isolate (D1) was reduced by 41-100% relative to locules inoculated with A. flavus alone. The severity of damage to locules inoculated simultaneously with A. flavus and with D1 was reduced by 60-l00% relative to locules inoculated with A. flavus alone. Isolate D1, identified as Pseudomonas cepacia, completely inhibited the growth of A. flavus on synthetic media.