Detection of Aspergillus fumigatus hyphae by solid phase cytometry

A rapid method based on solid phase cytometry (SPC) for the detection of Aspergillus fumigatus hyphae is described. With an enzymatic "viability" staining procedure, fungal hyphae can be detected non-specifically within the hour. By combining this procedure with an immunofluorescence labelling, a distinction between Aspergillus spp. and other clinically important fungi is possible, except for Penicillium spp. due to cross-reactivity. To differentiate both genera, microcolonies are generated by incubation at 45 degrees C prior to viability staining. The latter approach in conjunction with immunofluorescence labelling allows a quasi-specific detection of A. fumigatus hyphae and has shown its applicability to samples of bronchoalveolar lavage liquid (BAL).     

烟曲霉的细胞外囊泡蛋白质及RNA分析

目的 分析烟曲霉的细胞外囊泡(extracellular vesicles, EVs)中的重要成分,以进一步明确曲霉致病机制。方法 通过离心法分离烟曲霉的囊泡,用电镜观察形态。采用马尔文纳米颗粒跟踪分析仪分析溶液中EVs的大小分布。通过质谱仪对囊泡内处理后的肽段进行分析。二级质谱数据使用Maxquant (v1.5.2.8)进行检索。检测RNA分布,通过数据库分析烟曲霉EVs中miRNA可能参与的一些通路。结果 电镜下烟曲霉的EVs可见明显的双层脂质结构。NTA发现烟曲霉分析的EVs大小主要集中在130 nm左右。EVs蛋白中不稳定蛋白为9种(15%),其余为稳定蛋白,而等电点(isoelectric point, PI)>7的为6种蛋白。EVs中大部分是胞浆蛋白,其余比较多的是细胞外分泌蛋白,但仍有25%的蛋白不能定位。通过跨膜蛋白预测(transmembrane prediction, TMPred)和糖基磷脂酰肌醇(glycosylphosphatidylinositol, GPI)预测,有5种蛋白存在于双层脂质膜上的蛋白。检测到RNA中rRNA和tRNA分别占59.7%和...     

Inhibitory Effect of Aspergillus fumigatus Extract on matrix metalloproteinases Expression

BACKGROUND: Matrix metalloproteinases (MMPs) play a role in several physiologic and pathologic events. There are some evidence indicating the involvement of MMPs in the pathophysiology of fungal infections. METHODS: Here we study somatic extract of Aspergillus fumigatus. The influence of Aspergillus vs. two other fungal extracts on MMPs production by Fibrosarcoma cell line was investigated using an in vitro cytotoxicity assay, sodium dodecyl sulfate-polyacrylamide and gelatin zymography. RESULTS: Comparative dose-dependent inhibitory effects on MMPs were seen by A. fumigatus extract and compared with some steroidal and non-steroidal drugs. Cytotoxicity analysis of our extract revealed much lower cell death than other examined agents. CONCLUSIONS: Since inhibition of MMPs activity has been employed in modality therapy in such diseases as cancer, this extract might be promising in the preparation of anti-MMP therapeutic derivatives.     

Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.     

Distinct roles for intra- and extracellular siderophores during Aspergillus fumigatus infection

Siderophore biosynthesis by the highly lethal mould Aspergillus fumigatus is essential for virulence, but non-existent in humans, presenting a rare opportunity to strategize therapeutically against this pathogen. We have previously demonstrated that A. fumigatus excretes fusarinine C and triacetylfusarinine C to capture extracellular iron, and uses ferricrocin for hyphal iron storage. Here, we delineate pathways of intra- and extracellular siderophore biosynthesis and show that A. fumigatus synthesizes a developmentally regulated fourth siderophore, termed hydroxyferricrocin, employed for conidial iron storage. By inactivation of the nonribosomal peptide synthetase SidC, we demonstrate that the intracellular siderophores are required for germ tube formation, asexual sporulation, resistance to oxidative stress, catalase A activity, and virulence. Restoration of the conidial hydroxyferricrocin content partially rescues the virulence of the apathogenic siderophore null mutant Delta sidA, demonstrating an important role for the conidial siderophore during initiation of infection. Abrogation of extracellular siderophore biosynthesis following inactivation of the acyl transferase SidF or the nonribosomal peptide synthetase SidD leads to complete dependence upon reductive iron assimilation for growth under iron-limiting conditions, partial sensitivity to oxidative stress, and significantly reduced virulence, despite normal germ tube formation. Our findings reveal distinct cellular and disease-related roles for intra- and extracellular siderophores during mammalian Aspergillus infection.     

An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity

Invasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life-threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase-chain-reaction-based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS-polyacrylamide gel electrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild-type cells, the mutant had undetectable activity on azocollagen and elastin-Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.     

Detection of Aspergillus fumigatus hyphae in respiratory secretions by membrane filtration, fluorescent labelling and laser scanning

The detection of Aspergillus fumigatus hyphae in bronchoalveolar lavage fluid (BAL) and sputum is diagnostically useful in patients at risk of invasive aspergillosis. We report a dedicated enzymatic-chemical sample pretreatment that allows the application of a previously described solid phase cytometry (SPC) method for detection of A. fumigatus hyphae in sputum and BAL samples. Non-specific detection of fungal hyphae by SPC is based on a 'viability' staining using carboxyfluorescein diacetate. For a specific detection of A. fumigatus hyphae by SPC, viability staining is combined with a pre-incubation at 45 degrees C, immunofluorescent labelling and microscopic recognition of the characteristic hyphal morphology. Low numbers of A. fumigatus hyphae (2-10 hyphae/sample) have now been demonstrated in spiked sputum using the non-specific and specific staining in 2.5 and 8.5 h, respectively.     

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.     

Chemotherapy of Aspergillus fumigatus keratitis: An experimental study

An experimental Keratitis study of Aspergillus fumigatus was performed in 130 rabbits divided into 12 groups of ten animals each. Three antifungal drugs (myconazole, amphotericin B and pimaricin) were tested using two procedures (topical drops and subconjunctival injections) and two different concentrations (500 and 10 000 times the MIC). In each case, the drugs were applied every 3 h starting 14 h after inoculation. Miconazole was useful at 10 mg/ ml concentration by topical drops and subconjunctival injections, but was less useful at 5 mg/ ml. Amphotericin B was useful at 5 mg/ ml concentration by topical drops and less useful at 2 mg/ ml.No differences were found between the two concentrations by subconjunctival administration. Pimaricin was useful by topical drops at 50 mg/ ml concentration and less useful at 10 mg/ ml as well as by subconjunctival injections.     

Pseudomonas aeruginosa and their small diffusible extracellular molecules inhibit Aspergillus fumigatus biofilm formation

Aspergillus fumigatus is often isolated from the lungs of cystic fibrosis (CF) patients, but unlike in severely immunocompromised individuals, the mortality rates are low. This suggests that competition from bacteria within the CF lung may be inhibitory. The purpose of this study was to investigate how Pseudomonas aeruginosa influences A. fumigatus conidial germination and biofilm formation. Aspergillus fumigatus biofilm formation was inhibited by direct contact with P. aeruginosa, but had no effect on preformed biofilm. A secreted heat-stable soluble factor was also shown to exhibit biofilm inhibition. Coculture of P. aeruginosa quorum-sensing mutants (PAO1:ΔLasI, PAO1:ΔLasR) did not significantly inhibit A. fumigatus biofilms (52.6-58.8%) to the same extent as that of the PA01 wild type (22.9-30.1%), both by direct and by indirect interaction (P<0.001). Planktonic and sessile inhibition assays with a series of short carbon chain molecules (decanol, decanoic acid and dodecanol) demonstrated that these molecules could both inhibit and disrupt biofilms in a concentration-dependent manner. Overall, this suggests that small diffusible and heat-stable molecules may be responsible for the competitive inhibition of filamentous fungal growth in polymicrobial environments such as the CF lung.     

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.     

The Volatome of Aspergillus fumigatus

Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of Aspergillus fumigatus that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by A. fumigatusin vitro, and we show that volatile production depends on the nutritional environment. A. fumigatus produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-trans-bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by A. fumigatus. The present analysis of in vitro volatile production by A. fumigatus suggests that VOCs may be used in the diagnosis of infections caused by this fungus.     

Conidial hydrophobins of Aspergillus fumigatus

The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and DeltarodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. DeltarodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of DeltarodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the DeltarodA DeltarodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.     

Conidial Hydrophobins of Aspergillus fumigatus

The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and ΔrodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. ΔrodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of ΔrodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the ΔrodA ΔrodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

The pathobiology of Aspergillus fumigatus

Aspergillus fumigatus is the most prevalent airborne fungal pathogen in developed countries, and in immunocompromised patients causes a usually fatal invasive aspergillosis (IA). Understanding the pathobiology of this fungal species requires not only analysis of the putative fungal virulence factors that stimulate fungal growth and/or survival in the lung environment, but also knowledge of the immune factors containing A. fumigatus in the immunocompetent host that can be debilitated by immunosuppressive therapies, triggering IA. Although the incidence of IA has dramatically increased in recent years, progress in these areas has been limited and, as yet, a single, true virulence factor has not been identified and the mechanisms responsible for protective immunity against A. fumigatus have yet to be elucidated.