Antigenic profile of some typical and septate phialide-strains of Aspergillus fumigatus

Antigens from typical and septate phialide-strains of Aspergillus fumigatus, A. fumigatus var. ellipticus, A. fischeri, A. flavus and A. niger were analysed by fused rocket and two-dimensional immunoelectrophoresis. Depsite strain-to-strain variations, isolates of the two morphological forms of A. fumigatus showed a close similarity in their antigenic profile; the percentage of qualitative sharing ranged between 80% and 100%. The number of antigens shared by the isolates of A. fumigatus var. ellipticus and A. fischeri as compared with a typical A. fumigatus isolate was relatively high. By contrast isolates of A. flavus and A. niger, which are taxonomically not so closely related to A. fumigatus, revealed a qualitative sharing of only 16% and 8%, respectively. The number of common antigenically active components identified in culture filtrate antigens from the typical and septate phialide-strains of A. fumigatus was around 29. Two-dimensional electrophoresis with tandem and intermediate gel proved further that the two types of A. fumigatus isolates are antigenically homologous. Present findings also support the view that the antigenically active components of this pathogen are predominantly protein-glycoprotein in nature.     

Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples

In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.     

Sexual development and cryptic sexuality in fungi: insights from Aspergillus species

Major insights into sexual development and cryptic sexuality within filamentous fungi have been gained from investigations using Aspergillus species. Here, an overview is first given into sexual morphogenesis in the aspergilli, describing the different types of sexual structures formed and how their production is influenced by a variety of environmental and nutritional factors. It is argued that the formation of cleistothecia and accessory tissues, such as Hülle cells and sclerotia, should be viewed as two independent but co-ordinated developmental pathways. Next, a comprehensive survey of over 75 genes associated with sexual reproduction in the aspergilli is presented, including genes relating to mating and the development of cleistothecia, sclerotia and ascospores. Most of these genes have been identified from studies involving the homothallic Aspergillus nidulans, but an increasing number of studies have now in addition characterized 'sex-related' genes from the heterothallic species Aspergillus fumigatus and Aspergillus flavus. A schematic developmental genetic network is proposed showing the inter-relatedness between these genes. Finally, the discovery of sexual reproduction in certain Aspergillus species that were formerly considered to be strictly asexual is reviewed, and the importance of these findings for cryptic sexuality in the aspergilli as a whole is discussed.     

Molecular characterization of the food-borne fungus Neosartorya fischeri (Malloch and Cain).

The food-borne fungus Neosartorya fischeri, which is phenotypically related to the human opportunistic pathogen Aspergillus fumigatus, causes spoilage of heat-processed fruit products. Genomic methods were used to type N. fischeri strains and identify the genomic relationship between A. fumigatus and N. fischeri and between the different varieties of N. fischeri. EcoRI restriction fragment length polymorphism (RFLP) patterns obtained after ethidium bromide staining could differentiate most of N. fischeri var. glabra and N. fischeri var. spinosa strains. On the contrary, all N. fischeri var. fischeri strains tested exhibit the same RFLP pattern, which was similar to the A. fumigatus pattern. Similarly, Southern hybridization with a ribosomal probe showed some polymorphism between N. fischeri var. glabra and N. fischeri var. spinosa strains but could not distinguish between N. fischeri var. fischeri and A. fumigatus strains. By using the endonucleases EcoRI, HindIII, and BglII to generate Southern blot patterns with a fragment of the A. fumigatus gene coding for a 33-kDa protease, it was possible to differentiate N. fischeri var. fischeri from A. fumigatus. The difference between N. fischeri and A. fumigatus was confirmed by the use of moderately repetitive nonribosomal A. fumigatus sequences. These results are in agreement with previous studies that showed important infraspecific polymorphism within N. fischeri var. glabra and N. fischeri var. spinosa and, in contrast, the homogeneity of N. fischeri var. fischeri strains. A unique Southern blot pattern was seen for each strain of N. fischeri fingerprinted with the A. fumigatus repetitive sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH4)2SO4 treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100 degrees C) at pH 1.8, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

Variation of a 470 000 daltons antigen complex and catalase antigen in clinical isolates of Aspergillus fumigatus

Antigens in ruptured mycelium of 18 Aspergillus strains including 14 clinical isolates of A. fumigatus were studied by immunoelectrophoresis. One antigenic component of molecular weight 470 000 previously characterized by hydrophobic interaction chromatography and gel filtration and a second component with catalase activity were detected in all A. fumigatus isolates but in varying quantities. The 470 000 antigen complex cross-reacted with antigens in A. flavus and A. nidulans but not in A. niger or A. terreus. A. fumigatus catalase antigen cross-reacted with catalase in A. flavus, A. nidulans and A. terreus, but not in A. niger. One A. fumigatus isolate produced two catalase antigens showing a reaction of partial identity. A. flavus also produced two catalase antigens, one of which was species-specific.     

Aspergillus Spp. as an important occupational risk factor among susceptible individuals

The prevalence of different species of aspergilli was studied for two consecutive years (from April 1989 to March 1991) in a mechanised bakery, a poultry farm and a large municipal hospital in Delhi, India using an Andersen six stage volumetric sampler. A rich diversity of aspergilli (26 species) were recorded in each environment. The major contributors in each environment were Aspergillus flavus, A. niger, A. versicolor, A. sydowi, A. fumigatus, A. japonicus. The concentration of aspergilli was higher inside the work place compared to the outside air, with 25% to 45% contribution to the total fungal load. The concentration of A. flavus was higher in the storage section of the bakery and in the shed of the poultry farm, while A. niger was characteristic of the packing section and the hatchery. The hospital, which was naturally ventilated with a continuous mixing of indoor and outdoor air had a significantly high concentration of A. flavus (p < 0.05) inside the ward. Some of the frequently encountered species of Aspergillus are A. janus, A. ochraceous, A. nidulans, A. variecolor, A. luchuensis and A. terreus. Other species of Aspergillus had a very low occurrence, accounting for less than 1% of the total fungal load.     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH₄)₂SO₄ treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100C) at pH 18, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

G-protein and cAMP-mediated signaling in aspergilli: a genomic perspective

We have carried out an in silico exploration of the genomes of Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus oryzae, and identified components of G-protein/cAMP-mediated signaling. Putative G-protein coupled receptors (GPCRs) were distributed over nine classes. The GPCRs within classes were well conserved among aspergilli but varied in other ascomycetes. As previously observed in A. nidulans and other fungi, three Galpha, one Gbeta, and one Ggamma subunits of G proteins were identified in A. fumigatus, whereas an additional likely non-functional Galpha subunit was present in A. oryzae. While most fungal species had five proteins containing the regulator of G-protein signaling (RGS) domain predicted to participate in attenuation of G-protein signaling, A. fumigatus and A. oryzae had an additional RGS protein (RgsD) related to RgsA of A. nidulans. Genes encoding adenylate cyclase, a regulatory subunit and two catalytic subunits of the cAMP-dependent protein kinase, were also identified in the three aspergilli. Finally, regulators of cAMP signaling including low- and high-affinity phosphodiesterases were identified. Taken together, our data indicate a striking diversity at the GPCR level, but little diversity of components at the G-protein and cAMP-signaling level. This may reflect the abilities of these fungi to adapt to various ecological niches and to integrate diverse environmental cues into highly conserved cellular processes.     

Standardization and characterization of antigens for the diagnosis of aspergillosis

The aim of this study was to develop and characterize antigens for the diagnosis of aspergillosis. Nine strains of Aspergillus species Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger were grown in Sabouraud and Smith broth to produce exoantigens. The antigens were tested by immunodiffusion against sera from patients with aspergillosis and other systemic mycoses. The protein fraction of the antigens was detected by SDS-PAGE; Western blot and representative bands were assessed by mass spectrometry coupled to a nano Acquity UltraPerformance LC and analyzed by the Mascot search engine. Concurrently, all sera were tested with Platelia Aspergillus EIA. The most reactive antigens to sera from patients infected by A. fumigatus were produced by A. fumigatus MG2 Sabouraud and pooled A.?fumigatus Sabouraud samples, both with a sensitivity of 93% and specificity of 100% and 97%, respectively. Aspergillus niger and A. flavus antigens were reactive against A. niger and A. flavus sera, each one with a sensitivity and specificity of 100%. Two proteins, probably responsible for antigenic activity, β-glucosidase in A. fumigatus and α-amylase in A. niger were attained. The commercial kit had a specificity of 22%, sensitivity of 100%, positive predictive value of 48%, and negative predictive value of 100%. The antigens produced showed high sensitivity and specificity and can be exploited for diagnostics of aspergilloma.     

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.     

Specificity of antigens from pathogenic Aspergillus species. I. Studies with ELISA and immunofluorescence

Studies were made by enzyme linked immunosorbent assay (ELISA) and indirect fluorescent antibody (IFA) tests on the reactivities and specificities of 13 antigens prepared from four species of Aspergillus against antisera from immunized rabbits and 64 sera from patients with aspergillosis, other systemic mycoses and nocardiosis. Although reactions in both serological tests were invariably strongest with homologous antigen: antibody systems, antisera from rabbits immunized with A. fumigatus, Blastomyces dermatitidis, Candida albicans and Paracoccidioides brasiliensis reacted in the ELISA test with all of the Aspergillus antigens. In contrast, cross-reactivity was virtually non-existent with antiserum to Histoplasma capsulatum. Of five antigens prepared from A fumigatus tested by ELISA against human sera from patients with aspergillosis and other nocardial and systemic fungal infections, sensitivities varied from 81 to 100% for sera from 32 patients with aspergillosis, and specificities from 20 to 97% for sera from 30 patients with nocardiosis and other systemic mycoses. Purified A. fumigatus C antigen reacted weakly with sera from eight of these 30 patients, but the reactions were readily distinguishable from those obtained with sera from patients with aspergillosis. At optimal serum dilutions, cross-reactivities of A. fumigatus in the IFA studies were non-existent in the sera from 28 patients with candidosis, coccidioidomycosis, cryptococcosis, histoplasmosis, paracoccidioidomycosis and nocardiosis. Sensitivities of IFA were 94% for patients with aspergilloma and 83% for patients with allergic bronchopulmonary aspergillosis.     

Partial purification and characterization of mold antigens commonly found in foods.

Rapid methods are needed for detection of molds in foods; therefore, an enzyme-linked immunosorbent assay was developed. The extracellular and mycelial antigens for Mucor, Aspergillus, Cladosporium, and Geotrichum species were partially purified and characterized. The molecular masses of the mycelial and extracellular antigens, as determined by size exclusion chromatography, ranged from 4.5 x 10(5) to 6.7 x 10(5) Da. There was only one main antigenic peak separated by Sepharose CL-4B and concanavalin A-Sepharose columns for Mucor, Cladosporium, and Geotrichum mycelial and extracellular antigens, but there were two for Aspergillus mycelial antigens and three for Aspergillus extracellular antigens. These antigens contained 10 to 50% protein which was part of the active site since protease digestion significantly decreased antigenic activity. Neutral sugars, ranging from 13 to 75%, made up the rest of the active site, and < 1% phosphate was detected in mycelial antigens. Geotrichum, Cladosporium, and Aspergillus antigens contained mainly glucose, galactose, and mannose. Mucor antigens contained these sugars plus fucose. The percentage of sugars differed between the mycelia and extracellular antigens. Enzymatic digestion and competitive inhibition tests using different sugar derivatives showed that galactosyl residues with beta linkages were immunodominant for Aspergillus, Geotrichum, and Cladosporium antigens and mannosyl residues with alpha linkages were immunodominant for Mucor antigens.     

Natural occurrence of Aspergillus fumigatus in cane sugar mills.

The environmental distribution of Aspergillus fumigatus in 2 cane-sugar mills and one paper factory in northern India is compared with 2 localities in Delhi. The preponderance of the species at the U.D. Sugar Mills, Shamli, was contrary to its low prevalence in the University of Delhi campus and at Subzimandi, the vegetable and fruit market of Delhi. Aspergillus fumigatus accounted for 42.5% of the total aerial fungal colony counts recorded in the Shamli Mills as against 2% in Delhi. The predominant aerial fungus at Subzimandi was A. niger whereas aspergilli were overshelmingly outnumbered by other fungi in the University of Delhi campus. Within the Shamli Mills, the bagasse-containing sites had a significantly higher aerial prevalence (50.3%) of A. fumigatus than the bagasse-free sites (13.5%). Furthermore, A. fumigatus was more prevalent in the operational (57.2%) than in the non-operational period (23.8%) of the mills. The high frequency of isolations of A. fumigatus from and its dense population in sugar-cane bagasse seemed to suggest a special association of the fungus with this substrate.     

烟曲霉Asp f3和Asp f4线性B细胞抗原表位最小基序鉴定

烟曲霉(Aspergillus fumigatus)是一种广泛存在于自然界中的条件致病菌,其产生的分生孢子被易感人群吸入后定植于肺部,引起3种曲霉病:致咯血的曲霉肿、致肺纤维化的变应性支气管肺曲霉病、致较高死亡率的侵袭性曲霉病。目前临床上用于诊断烟曲霉感染的血清免疫学指标有半乳甘露聚糖和1,3-β-D-葡聚糖,但特异度和灵敏度方面存在一定的局限性。Asp f3和Asp f4为烟曲霉主要抗原,在感染者血清中存在相应的循环抗体。本研究分别用兔抗Asp f3和Asp f4抗血清对其进行抗原表位扫描,鉴定了8个Asp f3和6个Asp f4最小表位基序肽。用所鉴定的最小表位基序与烟曲霉其他抗原的最小表位基序构建嵌合肽,可能有助于提高烟曲霉感染诊断的特异度和灵敏度。     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.     

DNA sequence characterization and molecular evolution of MAT1 and MAT2 mating-type loci of the self-compatible ascomycete mold Neosartorya fischeri

Degenerate PCR and chromosome-walking approaches were used to identify mating-type (MAT) genes and flanking regions from the homothallic (sexually self-fertile) euascomycete fungus Neosartorya fischeri, a close relative of the opportunistic human pathogen Aspergillus fumigatus. Both putative alpha- and high-mobility-group-domain MAT genes were found within the same genome, providing a functional explanation for self-fertility. However, unlike those in many homothallic euascomycetes (Pezizomycotina), the genes were not found adjacent to each other and were termed MAT1 and MAT2 to recognize the presence of distinct loci. Complete copies of putative APN1 (DNA lyase) and SLA2 (cytoskeleton assembly control) genes were found bordering the MAT1 locus. Partial copies of APN1 and SLA2 were also found bordering the MAT2 locus, but these copies bore the genetic hallmarks of pseudogenes. Genome comparisons revealed synteny over at least 23,300 bp between the N. fischeri MAT1 region and the A. fumigatus MAT locus region, but no such long-range conservation in the N. fischeri MAT2 region was evident. The sequence upstream of MAT2 contained numerous candidate transposase genes. These results demonstrate a novel means involving the segmental translocation of a chromosomal region by which the ability to undergo self-fertilization may be acquired. The results are also discussed in relation to their significance in indicating that heterothallism may be ancestral within the Aspergillus section Fumigati.     

Specificity of antigens from pathogenic Aspergillus species. II. Studies with line immunoelectrophoresis

Somatic (mycelial) and metabolic (culture filtrate) antigens of Aspergillus flavus, A. fumigatus, A. nidulans, A. niger and A. terreus were compared by line immunoelectrophoresis with sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma, or from immunized animals. Number of lines observed when tested with human sera were similar for somatic and metabolic preparations of A. fumigatus, but up to 33 lines were present when both types of antigens were tested simultaneously. Cross-reactions between heterologous antigens and sera from patients with aspergilloma or ABPA were uncommon. In contrast, cross-reactions were common when standard antisera prepared in animals against heterologous species of Aspergillus were tested against A. fumigatus antigens. Lines of identity between homologous antigens and those from A. fumigatus were observed in 5 of 9 lines obtained with A. flavus, 4 of 16 lines of A. nidulans, 4 of 9 lines of A. niger and 4 of 8 lines of A. terreus.