Rapid identification of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> within the section <Emphasis Type="Italic">Fumigati</Emphasis>

Background  

New fungal species that are morphologically similar to Aspergillus fumigatus were recently described and included in section Fumigati. Misidentification of such fungal species, particularly of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs. Nevertheless, A. fumigatus still accounts for more than 90% of all invasive aspergillosis cases. The purpose of the present study was to develop a rapid method for the molecular identification of A. fumigatus to distinguish it from other species within the section Fumigati.     

Genetic variability within theAspergillus viridinutans species

The intraspecific variability of theAspergillus viridinutans species was examined using various techniques including morphological examinations, carbon-source-utilization tests, restriction-enzyme analysis of the mitochondrial and nuclear DNA, and sequence analysis of part of the β-tubulin gene. Although the genetic distances between the isolates were higher than between,e.g., A. fumigatus andNeosartorya fischeri, mostA. viridinutans isolates, together withN. aureola andN. udagawae strains, were found to belong to a single cluster based on sequence data. Strain FRR 1266, which was earlier classified as a highly divergentA. fumigatus isolate, was found to belong to theA. viridinutans species. The ochratoxin A producingA. viridinutans strain IMI 306 135 was most closely related to an asexual isolate. These two latter strains were more closely related toA. fumigatus andN. fischeri than to anyA. viridinutans strains, and possibly represent a new species in theAspergillus sectionFumigati. The dendrogram based on carbon source utilization data and results of restriction analysis of the mitochondrial and nuclear DNA of the strains supported most of the evolutionary relationships observed on the basis of sequence data. The results indicate that the presence or absence of nodding conidial heads is not an unequivocal morphological character for the identification of species within theAspergillus sectionFumigati.     

Determination of Azole Resistance and TR<Subscript>34</Subscript>/L98H Mutations in Isolates of <Emphasis Type="Italic">Aspergillus</Emphasis> Section <Emphasis Type="Italic">Fumigati</Emphasis> from Turkish Cystic Fibrosis Patients

Background

Aspergillus fumigatus is the species section Fumigati most frequently isolated from the respiratory tract of cystic fibrosis (CF) patients. Recent studies suggest that mutations in the Cyp51 gene, particularly TR₃₄/L98H, are responsible for azole resistance.

Objectives and Methods

The focus of this study was on section Fumigati isolates isolated from the respiratory tract samples of CF patients. More specifically, the goal was to detect A. fumigatus isolates, test their antifungal susceptibility to itraconazole, voriconazole and posaconazole, and finally determine the presence of TR₃₄/L98H and other mutations in the isolates Cyp51A gene.

Results and Conclusions

A set of 31 isolates of Aspergillus section Fumigati were obtained from the sputum samples of 6 CF patients and subsequently identified to species level by microsatellite genotyping. All isolates were determined as A. fumigatus and involved 14 different genotypes. The minimal inhibitory concentrations to the three azoles were determined by the E-test method, and the Cyp51A gene was sequenced. One of the genotypes was found to be resistant to all azoles but no mutations were detected in the Cyp51A gene, especially the TR₃₄/L98H mutation. Therefore, mutations in genes other than Cyp51A or other distinct mechanisms may be responsible for this reported multiazole resistance found in a Turkish CF patient.

     

Phylogenetic analysis of newly described Neosartorya species

Isolates representing newly described Neosartorya species, and isolates with abnormal morphologies from Aspergillus section Fumigati were examined by phylogenetic analysis of sequences of part of their -tubulin gene. Phylogenetic analyses supported the earlier suggestions that heterothallism is a derived character, and that sexuality was lost several times during the evolution of Aspergillus section Fumigati. The heterothallic N. fennelliae and N. udagawae strains were found to be closely related to the homothallic Neosartorya sp. NRRL 4179 and N. aureola, respectively. Aspergillus sp. FRR 1266, which was earlier described as a variant of A. fumigatus, was found to be closely related to A. viridinutans. Another abnormal asexual isolate was found to be closely related to A. fumigatus and N. fischeri. Phylogenetic relationships among newly described Neosartorya species and other taxa were successfully established based on phylogenetic analysis of -tubulin sequences.     

Efficacy of the Combination of Voriconazole and Caspofungin in Experimental Pulmonary Aspergillosis by Different Aspergillus Species

Objectives

Invasive pulmonary aspergillosis (IPA) caused by Aspergillus fumigatus, Aspergillus flavus, or Aspergillus niger is associated with high mortality. We evaluated the efficacy and compared the therapeutic effect differences of voriconazole (VRC) in combination with caspofungin (CAS) in transiently neutropenic rats infected by A. fumigatus, A. flavus, or A. niger.

Methods

Treatment groups consisted of VRC (10 mg/kg q12 h) monotherapy, CAS (1 mg/kg/day) monotherapy, combination of VRC (10 mg/kg q12 h) + CAS (1 mg/kg/day), and no drug for 10 consecutive days. The efficacy and the difference in the treatments were evaluated through prolongation of survival, reduction in serum galactomannan levels and residual fungal burden, and histological studies.

Results

For all the strains, the combination of VRC and CAS led to significant prolongation in survival (P < 0.05) and reduction in residual fungal burden (P < 0.05) compared with CAS alone, and decrease in serum galactomannan levels (P < 0.05) compared with either agent alone. The survival in the combined therapy groups was significantly improved compared to VRC monotherapy for the strains of A. flavus and A. niger (P < 0.05), but no significant difference for the strains of A. fumigatus (P > 0.05).

Conclusions

Combination of VRC and CAS was synergistic in IPA by A. flavus and A. niger, but small efficacy benefits in IPA by A. fumigatus.     

Re-identification of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> sensu lato based on a new concept of species delimitation

The species concept of Aspergillus fumigatus sensu stricto has recently been defined by polyphasic taxonomy. Based on the new concept of species delimitations, 146 worldwide strains of Aspergillus fumigatus sensu lato were re-identified. Of those 146 strains, 140 (95.8%) could be identified as A. fumigatus sensu stricto, 3 (2.1%) as A. lentulus, and the remaining 3 strains as A. viridinutans complex, Neosartorya udagawae, and N. cf. nishimurae. Of 98 clinical strains, only 1 from dolphin nostril was identified as A. lentulus and not A. fumigatus sensu stricto. Random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers PELF and URP1F produced nearly the same band patterns among 136 strains of A. fumigatus sensu stricto while discriminated the species from its related species. We also discussed about identification of several atypical A. fumigatus strains from clinical environments.     

Aspergillus siamensis sp. nov. from soil in Thailand

A new species of Aspergillus section Fumigati, Aspergillus siamensis, isolated from coastal forest soil in Samaesarn island, Chonburi province, Eastern Thailand, is described and illustrated. This species is characterized by its broadly lenticular ascospores with two wide equatorial crests and finely spinulose and rugose convex surfaces and produced pale pinkish exudates after 14 d incubation on Czapek agar. The validation of this new species was supported further by analyses of the β-tubulin, calmodulin and actin gene sequences.     

Disparate Proteome Responses of Pathogenic and Nonpathogenic Aspergilli to Human Serum Measured by Activity-Based Protein Profiling (ABPP)

Aspergillus fumigatus is the primary pathogen causing the devastating pulmonary disease Invasive Aspergillosis in immunocompromised individuals. There is high genomic synteny between A. fumigatus and closely related rarely pathogenic Neosartorya fischeri and Aspergillus clavatus genomes. We applied activity-based protein profiling to compare unique or overexpressed activity-based probe-reactive proteins of all three fungi over time in minimal media growth and in response to human serum. We found 360 probe-reactive proteins exclusive to A. fumigatus, including known virulence associated proteins, and 13 proteins associated with stress response exclusive to A. fumigatus culture in serum. Though the fungi are highly orthologous, A. fumigatus has a significantly greater number of ABP-reactive proteins across varied biological process. Only 50% of expected orthologs of measured A. fumigatus reactive proteins were observed in N. fischeri and A. clavatus. Activity-based protein profiling identified a number of processes that were induced by human serum in A. fumigatus relative to N. fischeri and A. clavatus. These included actin organization and assembly, transport, and fatty acid, cell membrane, and cell wall synthesis. Additionally, signaling proteins regulating vegetative growth, conidiation, and cell wall integrity, required for appropriate cellular response to external stimuli, had higher activity-based probe-protein reaction over time in A. fumigatus and N. fisheri, but not in A. clavatus. Together, we show that measured proteins and physiological processes identified solely or significantly over-represented in A. fumigatus reveal a unique adaptive response to human protein not found in closely related, but rarely pathogenic aspergilli. These unique activity-based probe-protein responses to culture condition may reveal how A. fumigatus initiates pulmonary invasion leading to Invasive Aspergillosis.Invasive aspergillosis (IA)¹ is a devastating infection caused by the ubiquitous saprophytic filamentous fungus Aspergillus fumigatus (Af) (1). Af is an opportunistic pathogen with no true virulence factors. Its pathogenicity is often attributed to its thermotolerance, response to oxidative stress, ability to grow in hypoxic or iron limiting environments, and its ability to use a variety of carbon and nitrogen sources as nutrients, such as proteins derived from the human host (2). A thorough understanding of biological processes or factors that facilitate pathogenic Af infection compared with other microbial infections is needed to assist treatment and diagnosis of IA.Af protein activity regulation and function, attenuated by environmental response and adaptation, is critical for opportunistic infection and development of IA (3). Activity-based protein profiling (ABPP), coupled to mass spectrometry (MS), is a powerful chemical biology approach for directly identifying a subset of proteins (4). ABPP employs activity-based probes (ABPs) to covalently label and enrich functional families of proteins, thereby reducing the complexity of a proteome, and facilitating observation of low abundance proteins (4). The approach is uniquely suited for studying the ABP-reactive proteome response to human protein by Af when compared with related, but rarely pathogenic Neosartorya fischeri (Nf) and Aspergillus clavatus (Ac).In a recent study, we used ABPP in combination with a MS-based approach to investigate ABP-protein reactivity of Af (5). We multiplexed, for simultaneous measurement, our click chemistry compatible general cysteine reactive vinyl sulfonate ester (VSE-1) and serine hydrolase specific fluorophosphonate (Fp-2) ABPs to analyze the ABP-reactive Af proteome of mycelia cultured in the presence and absence of human serum (HS) (5). Comparison of probe-labeled samples to nonprobe labeled global samples revealed that VSE-1 and Fp-2 enriched a defined subset of proteins. Hence, we identified a series of distinct responses of Af to HS.Herein, we hypothesize that Af uniquely reacts to HS when compared with rarely pathogenic Nf and Ac (6–9). Fedorova et al. found that the three species share a core of 7514 genes, and that only 8.5%, 13.5%, and 12.6% of the Af, Nf, and Ac genomes are organism specific (6). The high percentage of genomic overlap, which includes virulence associated genes, suggests that IA infection may be caused by protein regulation and function in response to environment (10, 11). Therefore, we have employed ABPP coupled to MS to identify ABP-reactive proteins and associated pathways in Af dramatically over- or under-represented in the other aspergilli in the presence of HS. We believe that unique and/or overexpressed functional pathways may be the true virulence “factor” of Af, by enabling adaptation to environmental stress and deployment of requisite metabolic regulation to survive and replicate in the host environment.     

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection,Identification and Genotyping in Clinical Specimens

Objective

Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.

Methods

To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.

Results

A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.

Conclusions

The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.     

Aspergillus yunnanensis,a new and rare species in the Aspergillus section Jani

Aspergillus is a monophyletic genus comprising the subgenera Aspergillus, Circumdati, Cremei, Fumigati, Nidulantes and Polypaecilum. The subgenus Circumdati contains many economically important species and mycotoxin producers. Section Jani was recently introduced with morphological and molecular support. In the present study, two strains isolated from farmland soil were assigned in section Jani based on multi-locus phylogenetic analyses but showed low similarity with existing species. Further morphological observation found they had wider vesicles and conidia connections which were different from the known species. Based on phylogenetic and morphological data, Aspergillus yunnanensis was introduced as the third species in section Jani. Members in section Jani are rarely distributed, this is the first report of this section in China.     

New resources for functional analysis of omics data for the genus <Emphasis Type="Italic">Aspergillus</Emphasis>

Background

Detailed and comprehensive genome annotation can be considered a prerequisite for effective analysis and interpretation of omics data. As such, Gene Ontology (GO) annotation has become a well accepted framework for functional annotation. The genus Aspergillus comprises fungal species that are important model organisms, plant and human pathogens as well as industrial workhorses. However, GO annotation based on both computational predictions and extended manual curation has so far only been available for one of its species, namely A. nidulans.

Results

Based on protein homology, we mapped 97% of the 3,498 GO annotated A. nidulans genes to at least one of seven other Aspergillus species: A. niger, A. fumigatus, A. flavus, A. clavatus, A. terreus, A. oryzae and Neosartorya fischeri. GO annotation files compatible with diverse publicly available tools have been generated and deposited online. To further improve their accessibility, we developed a web application for GO enrichment analysis named FetGOat and integrated GO annotations for all Aspergillus species with public genome sequences. Both the annotation files and the web application FetGOat are accessible via the Broad Institute's website (http://www.broadinstitute.org/fetgoat/index.html). To demonstrate the value of those new resources for functional analysis of omics data for the genus Aspergillus, we performed two case studies analyzing microarray data recently published for A. nidulans, A. niger and A. oryzae.

Conclusions

We mapped A. nidulans GO annotation to seven other Aspergilli. By depositing the newly mapped GO annotation online as well as integrating it into the web tool FetGOat, we provide new, valuable and easily accessible resources for omics data analysis and interpretation for the genus Aspergillus. Furthermore, we have given a general example of how a well annotated genome can help improving GO annotation of related species to subsequently facilitate the interpretation of omics data.

     

Carbohydrate Specificity of Antibodies against Phytopathogenic Fungi of the <Emphasis Type="Italic">Aspergillus</Emphasis> Genus

Brush rabbits were immunized with injections prepared from the fungi Aspergillus fumigatus, Aspergillus niger, and Aspergillus repens. A library of synthetic biotinylated oligosaccharides containing the key fragments of antigenic polysaccharides of the fungal cell wall—galctomannan, α- and β-glucans, mannan, and chitin—was used to analyze carbohydrate specificity. The anticarbohydrate antibodies obtained from animals immunized with preparations from A. fumigatus and A. repens predominantly recognized epitopes containing galactofuranoside residues, while the majority of the antibodies against A. niger bound the chitooligosaccharide ligand. These results are the basis for the identification of specific markers required for the development of immunoenzyme test systems.     

Sexualidad y patogenicidad en Aspergillus fumigatus: ¿existe alguna relación?

BackgroundAspergillus fumigatus, like many other fungal species of clinical relevance, has been traditionally regarded as an asexual organism. However, in last few years several pieces of evidence question this such assumption, suggesting that the sexual state of A. fumigatus may still be undiscovered. These investigations have finally led to the recent discovery of a teleomorph stage of A. fumigatus, which has been named Neosartorya fumigata.AimsTo review the most important findings on A. fumigatus sexuality and discuss the possible implications of such findings on its pathogenicity.MethodsA bibliographic search was performed to find the main works that study the sexuality of fungal pathogens and, especially, of A. fumigatus. Moreover, data from our recent investigations in this field were also introduced to the discussion.ResultsThe existence of a teleomorph for A. fumigatus could have significant clinical repercussions, as sexual reproduction might produce offspring with increased virulence and/or resistance to antifungal agents. In this sense, the results of our investigations suggest the existence of an association between the MAT1-1 mating type and the invasiveness of A. fumigatus isolates.ConclusionsThe study of the sexual reproduction of the fungal pathogens and its possible relationship with virulence will continue to be a topic of interest during the next years, not only because of its basic interest, but also for the possible clinical repercussions.     

Comparison of Chemical Sensitivity of Fresh and Long-Stored Heat Resistant Neosartorya fischeri Environmental Isolates Using BIOLOG Phenotype MicroArray System

Spoilage of heat processed food and beverage by heat resistant fungi (HRF) is a major problem for food industry in many countries. Neosartorya fischeri is the leading source of spoilage in thermally processed products. Its resistance to heat processing and toxigenicity makes studies about Neosartorya fischeri metabolism and chemical sensitivity essential. In this study chemical sensitivity of two environmental Neosartorya fischeri isolates were compared. One was isolated from canned apples in 1923 (DSM3700), the other from thermal processed strawberry product in 2012 ({"type":"entrez-nucleotide","attrs":{"text":"KC179765","term_id":"452916796"}}KC179765), used as long-stored and fresh isolate, respectively. The study was conducted using Biolog Phenotype MicroArray platforms of chemical sensitivity panel and traditional hole-plate method. The study allowed for obtaining data about Neosartorya fischeri growth inhibitors. The fresh isolate appeared to be much more resistant to chemical agents than the long-stored isolate. Based on phenotype microarray assay nitrogen compounds, toxic cations and membrane function compounds were the most effective in growth inhibition of N. fischeri isolates. According to the study zaragozic acid A, thallium(I) acetate and sodium selenate were potent and promising N. fischeri oriented fungicides which was confirmed by both chemical sensitivity microplates panel and traditional hole-plate methods.     

Antifungal activity of redox-active benzaldehydes that target cellular antioxidation

Background

Disruption of cellular antioxidation systems should be an effective method for control of fungal pathogens. Such disruption can be achieved with redox-active compounds. Natural phenolic compounds can serve as potent redox cyclers that inhibit microbial growth through destabilization of cellular redox homeostasis and/or antioxidation systems. The aim of this study was to identify benzaldehydes that disrupt the fungal antioxidation system. These compounds could then function as chemosensitizing agents in concert with conventional drugs or fungicides to improve antifungal efficacy.

Methods

Benzaldehydes were tested as natural antifungal agents against strains of Aspergillus fumigatus, A. flavus, A. terreus and Penicillium expansum, fungi that are causative agents of human invasive aspergillosis and/or are mycotoxigenic. The yeast Saccharomyces cerevisiae was also used as a model system for identifying gene targets of benzaldehydes. The efficacy of screened compounds as effective chemosensitizers or as antifungal agents in formulations was tested with methods outlined by the Clinical Laboratory Standards Institute (CLSI).

Results

Several benzaldehydes are identified having potent antifungal activity. Structure-activity analysis reveals that antifungal activity increases by the presence of an ortho-hydroxyl group in the aromatic ring. Use of deletion mutants in the oxidative stress-response pathway of S. cerevisiae (sod1Δ, sod2Δ, glr1Δ) and two mitogen-activated protein kinase (MAPK) mutants of A. fumigatus (sakAΔ, mpkCΔ), indicates antifungal activity of the benzaldehydes is through disruption of cellular antioxidation. Certain benzaldehydes, in combination with phenylpyrroles, overcome tolerance of A. fumigatus MAPK mutants to this agent and/or increase sensitivity of fungal pathogens to mitochondrial respiration inhibitory agents. Synergistic chemosensitization greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations. Effective inhibition of fungal growth can also be achieved using combinations of these benzaldehydes.

Conclusions

Natural benzaldehydes targeting cellular antioxidation components of fungi, such as superoxide dismutases, glutathione reductase, etc., effectively inhibit fungal growth. They possess antifungal or chemosensitizing capacity to enhance efficacy of conventional antifungal agents. Chemosensitization can reduce costs, abate resistance, and alleviate negative side effects associated with current antifungal treatments.     

Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP)

A novel 6.6 kDa antifungal peptide (NFAP) from the culture supernatant of the mold, Neosartorya fischeri (anamorf: Aspergillus fischerianus), and its encoding gene were isolated in this study. NFAP is a small, basic and cysteine-rich protein consisting of 57 amino acid residues. It shows 37.9-50% homology to similar proteins described in literature from Aspergillus clavatus, Aspergillus giganteus, Aspergillus niger, and Penicillium chrysogenum. The in silico presumed tertiary structure of NFAP, e.g. the presence of five antiparallel β-sheet connected with filaments, and stabilized by three disulfide bridges, is very similar to those of the defensin-like molecules. NFAP exhibited growth inhibitory action against filamentous fungi in a dose-dependent manner, and maintained high antifungal activity within broad pH and temperature ranges. Furthermore, it exhibited relevant resistance to proteolysis. All these characteristics make NFAP a promising candidate for further in vitro and in vivo investigations aiming at the development of new antifungal compounds.     

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0–6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn²⁺. The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K _m, V _max, K _cat, and K _cat/K _m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.