BACKGROUND: Previous research demonstrated that a homozygous mutation of g.136372044G>A (S12N) in caspase recruitment domain family member 9 ( CARD9 ) is critical for producing Aspergillus fumigatus -induced ( Af -induced) T helper 2 (T H 2)-mediated responses in allergic bronchopulmonary aspergillosis (ABPA). However, it remains unclear whether the CARD9S12N mutation, especially the heterozygous occurrence, predisposes the host to ABPA. METHODS: A total of 61 ABPA patients and 264 controls (including 156 healthy controls and 108 asthma patients) were recruited for sequencing the CARD9 locus to clarify whether patients with this heterozygous single-nucleotide polymorphisms are predisposed to the development of ABPA. A series of in vivo and in vitro experiments, such as quantitative real-time polymerase chain reaction, flow cytometry, and RNA isolation and quantification, were used to illuminate the involved mechanism of the disease. RESULTS: The presence of the p.S12N mutation was associated with a significant risk of ABPA in ABPA patients when compared with healthy controls and asthma patients, regardless of Aspergillus sensitivity. Relative to healthy controls without relevant allergies, the mutation of p.S12N was associated with a significant risk of ABPA (OR: 2.69 and 4.17 for GA and AA genotypes, P = 0.003 and 0.029, respectively). Compared with patients with asthma, ABPA patients had a significantly higher heterozygous mutation (GA genotype), indicating that p.S12N might be a significant ABPA-susceptibility locus ( aspergillus sensitized asthma: OR: 3.02, P = 0.009; aspergillus unsensitized asthma: OR: 2.94, P = 0.005). The mutant allele was preferentially expressed in ABPA patients with heterozygous CARD9S12N , which contributes to its functional alterations to facilitate Af -induced T H 2-mediated ABPA development. In terms of mechanism, Card9 wild-type ( Card9WT ) expression levels decreased significantly due to Af -induced decay of its messenger RNA compared to the heterozygous Card9S12N . In addition, ABPA patients with heterozygous CARD9S12N had increased Af -induced interleukin-5 production. CONCLUSION Our study provides the genetic evidence showing that the heterozygous mutation of CARD9S12N , followed by allele expression imbalance of CARD9S12N , facilitates the development of ABPA.
Humans ; Aspergillosis, Allergic Bronchopulmonary/complications* ; Aspergillus fumigatus/genetics* ; Asthma/genetics* ; Aspergillus ; Mutation/genetics* ; CARD Signaling Adaptor Proteins/genetics*

BACKGROUNDGlucocorticoid is speculated to be able to have Aspergillus fumigatus (A. fumigatus) being more susceptible to reactive oxygen species (ROS) by inhibiting Afyap1, the transcription factor activating protein-1 (AP-1) homologue in A. fumigatus, which may provide a clue to expand the clinical use of glucocorticoid in patients with fungal infections. In this study, we used dexamethasone to determine the direct effect on oxidative killing susceptibility of A. fumigatus in vitro, as well as the expression level of Afyap1 gene and its target genes (catalase and superoxide dismutase (SOD) genes).

METHODSA. fumigatus spores were treated with different concentrations (0, 0.02, 0.2 mg/ml) of glucocorticoids and assigned to four groups (A: 0.5 hour, B: 2 hours, C: 7 hours, D: 16 hours) according to the time of treatment. The H2O2 oxidative killing assay was done, using the standard method-spot test, in each group of A. fumigatus. We measured the oxidative killing susceptibility as well as the expression level of the gene Afyap1, CATA, SOD1 and SOD2 in A. fumigatus at each group. The antifungal susceptibility to itraconazole and amphotericin B in each group of A. fumigatus was also measured with M38-A2 method.

RESULTSThe oxidative killing susceptibility of A. fumigatus was increased, consistent with the reduction of Afyap1, CATA, SOD1 and SOD2 gene expression level after being treated with dexamethasone for 0.5 hours. However, these observations were disappeared along with being treated for longer time. The antifungal susceptibility to itraconazole and amphotericin B in the A. fumigatus strains treated with dexamethasone indicated no change, compared with those without dexamethasone treatment.

CONCLUSIONDexamethasone can have A. fumigatus being more susceptible to ROS when treated for shorter period (0.5 to 2 hours) via the reduction of Afyap1 gene expression as well as the down-stream enzyme-coding gene expression.

Aspergillus fumigatus ; drug effects ; genetics ; metabolism ; Dexamethasone ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Hydrogen Peroxide ; pharmacology

BACKGROUNDThe efficacies of current treatments for invasive aspergillus (IA) are unsatisfactory and new therapeutic targets or regimens to treat IA are urgently needed. Previous studies have indicated that the ability of conidia to invade host cells is critical in IA development and fibronectin has a hand in the conidia adherence process. In the clinical setting, many patients who receive glucocorticoid for extended periods are susceptible to Aspergillus fumigatus (A. fumigatus) infection, for this reason we investigated the effect of glucocorticoid on conidia invasiveness by comparing the invasiveness of A. fumigatus conidia in the type II human alveolar cell line (A549) cultured with different concentrations of dexamethasone. We also explored the relationships between dexamethasone and fibronectin expression.

METHODSFollowing culture with anti-fibronectin antibodies and/or dexamethasone, type II human alveolar A549 cells were infected with conidia of A. fumigatus. After 4 hours, the extracellular free conidia were washed away and the remaining immobilized conidia were released using Triton-X 100 and quantified by counting the colony-forming units. The invasiveness of conidia was measured by calculating the invasion rate (%). The transcription of the fibronectin gene in cells cultured with different concentrations of dexamethasone for 24 hours was tested by fluorogenic quantitative RT-PCR while the expression of fibronectinin cells cultured for 48 hours was tested by Western blotting and immunocytochemistry.

RESULTSA significant reduction in the invasiveness of conidia was seen in the cells cultured with anti-fibronectin antibody ((14.42 ± 1.68)% vs. (19.17 ± 2.53)%, P < 0.05), but no significant difference was observed in cells cultured with a combination of anti-fibronectin antibody and dexamethasone (6.37 ± 10(-5) mol/L). There was no correlation between the dexamethasone concentration and the invasiveness of conidia after dexamethasone pretreatment of cells for 4 hours. In contrast, after pretreated for 24 hours, the invasiveness of conidia in the presence of 6.37×10(-5) mol/L dexamethasone ((24.66 ± 2.41)%) was higher than for the control ((19.17 ± 2.53)%) and the 0.25×10(-5) mol/L group ((19.93 ± 3.06)%), and the invasiveness in the 1.27×10(-5) mol/L group ((22.47 ± 2.46)%) was also higher than in the control, P < 0.05. The relative transcripts of the fibronectin gene after exposure to 6.37×10(-5) mol/L dexamethasone (9.19×10(-3)±1.2×10(-3)) was higher than for the control (4.61×10(-3)± 1.54×10(-3)) and the 0.25×10(-5) mol/L group (6.20×10(-3)± 1.93×10(-3)), and expression in the 1.27×10(-5) mol/L group (7.94×10(-3)± 2.24×10(-3)) was also higher than for the control, P < 0.05. High concentrations of dexamethasone promoted fibronectin production after culture for 48 hours.

CONCLUSIONSDexamethasone can increase invasiveness of A. fumigatus conidia by promoting fibronectin expression. This may partially explain why patients who are given large doses of glucocorticoids for extended periods are more susceptible to A. fumigatus infection.

Aspergillus fumigatus ; pathogenicity ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Fibronectins ; genetics ; metabolism ; Gene Expression ; drug effects ; Humans

Exploring new beta-glucosidase genes is of great importance to industrialize beta-glucosidase. The genomes of Aspergillus fumigatus contain a bgl gene, which encodes a 65 kDa putative beta-glucosidase. The bgl gene was cloned into an expression plasmid and transformed to Escherichia coli BL21 (DE3). The bgl was expressed upon induction of Isopropyl beta-D-1-thiogalactopyranoside (IPTG). The recombinant protein was purified by GST-tag affinity chromatography. The purified recombinant Bgl was characterized using Esculin as substrate. The optimum temperature and pH were 45 degrees C and 5.0-6.0, respectively. The K(m) for Esculin was 17.7 mmol/L. The enzyme was stable in the range of pH 4-7. After incubation at 70 degrees C for 2 h, the recombinant Bgl remained 60% of its activity. Metal ions and chemical reagents had different influences on the activity of beta-glucosidase. Ca2+ (1 mmol/L) could increase enzyme activity slightly. On the contrary, the enzyme activity was greatly inhibited by 5 mmol/L Sodium dodecyl sulfate (SDS). Based on our results, the A. fumigatus Bgl was thermostable beta-glucosidase.
Aspergillus fumigatus ; enzymology ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; beta-Glucosidase ; biosynthesis ; genetics ; metabolism

Aspergillosis ; diagnosis ; microbiology ; Aspergillus fumigatus ; genetics ; isolation & purification ; DNA, Fungal ; genetics ; isolation & purification ; Dermatomycoses ; diagnosis ; microbiology ; Eye Diseases ; diagnosis ; microbiology ; Female ; Humans ; Middle Aged ; Polymerase Chain Reaction

OBJECTIVETo evaluate the feasibility of PCR/reverse line blot hybridization (RLB) assay in the detection and identification of clinical pathogens in fungal sinusitis (FS).

METHODSTwenty-six formalin-fixed and paraffin-embedded tissues and 8 fresh tissues of FS were collected from Beijing Tongren Hospital, Capital Medical University from May 2009 to February 2010. Pathological examination, fungal culture and ITS2 region sequencing were carried out. The DNA of all samples was extracted by standard procedure and fungal universal primers ITS3 and ITS4 were used for PCR amplification of all tissues. Then the amplified products were used for RLB with five fungal species-specific probes. The results of PCR/RLB were compared with ITS region sequencing, fungal culture and pathological examination.

RESULTSFor the biopsy tissues, fungal cultures were positive in 14 cases (41.2%); pathologic examination demonstrated fungal hyphae in all cases; ITS2 region sequencing was successful in 16 cases (47.1%); PCR/RLB showed A. flavus in 14 cases, A. fumigatus in 10 cases, A. niger in four cases, A. nidulans in one case, A. flavus and A. fumigatus in three cases, and A. fumigatus and A. niger in two cases.

CONCLUSIONSThe PCR/RLB assay is suitable for rapid and accurate detection and identification of the pathogenic fungus of FS. Compared with the conventional fungal culture and microscopy, pathologic examination and DNA sequencing, the PCR/RLB has the advantages of more economy, time saving, and higher sensitivity, specificity and throughput.

Aspergillus ; classification ; genetics ; isolation & purification ; Aspergillus flavus ; genetics ; isolation & purification ; Aspergillus fumigatus ; genetics ; isolation & purification ; Aspergillus niger ; genetics ; isolation & purification ; DNA Primers ; DNA, Fungal ; genetics ; Humans ; Mycoses ; diagnosis ; microbiology ; Nucleic Acid Hybridization ; methods ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sinusitis ; diagnosis ; microbiology

OBJECTIVETo differentiate between Aspergillus species and Mucorales of fungal sinusitis by immunohistochemistry.

METHODSFormalin-fixed paraffin-embedded tissue blocks of 66 cases of fungal sinusitis were retrieved from the archival files of Department of Pathology of Beijing Tongren Hospital during the period from 2001 to 2006. The samples included 29 cases of fungal balls, 12 cases of allergic fungal sinusitis, 24 cases of chronic invasive fungal sinusitis and 1 case of acute invasive fungal sinusitis. The types of fungi were 44 Aspergillus species (31 cases of A. fumigatus, 7 cases of A. flavus and 6 cases of A. terreus) and 22 Mucorales (14 cases of Mucor species and 8 cases of Rhizopus species). Immunohistochemistry was performed with MUC2, MUC5AC and MUC5B antibodies. The results were compared with histochemical study for periodic acid-Schiff (PAS) and Grocott methenamine silver (GMS) stains.

RESULTSImmunohistochemical study for MUC5B showed that the positive rate of Aspergillus species was 90.9%, in contrast to 4.5% in Mucorales (P < 0.001). The expression of MUC2 and MUC5AC was completely negative, whereas PAS and GMS stains were positive in all cases.

CONCLUSIONMUC5B antibody appears to be a useful immunohistochemical marker for identifying fungal types in tissue sections, especially in distinguishing between Aspergillus species and Mucorales in fungal sinusitis.

Antibodies, Fungal ; immunology ; Antibody Specificity ; immunology ; Aspergillosis ; diagnosis ; immunology ; Aspergillus flavus ; immunology ; Aspergillus fumigatus ; immunology ; Diagnosis, Differential ; Humans ; Immunohistochemistry ; methods ; Mucin-5B ; genetics ; immunology ; Mucor ; immunology ; Mycoses ; diagnosis ; immunology ; microbiology ; Sinusitis ; diagnosis ; microbiology

BACKGROUNDDuring the past decades, the incidence of invasive aspergillosis (IA) caused by Aspergillus fumigatus has increased dramatically. The aims of this study were to investigate the susceptibility of clinical isolates of A. fumigatus to triazole and the underlying cyp51A mutations in triazole-resistant A. fumigatus.

METHODSA total of 126 A. fumigatus clinical isolates from 126 patients with proven or probable IA were obtained from four large tertiary hospitals in Nanjing, China, between August 2012 and July 2015. The determination of minimal inhibitory concentrations (MICs) for itraconazole, voriconazole, and posaconazole was performed by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing reference method.

RESULTSA total of 4 A. fumigatus isolates (3.17%) were confirmed to be itraconazole resistant, with MICs of ≥8 mg/L, and one isolate (0.8%) was confirmed to be voriconazole resistant and posaconazole resistant, with MICs of 4 mg/L and 0.5 mg/L, respectively. We found that two of the 4 isolates of triazole-resistant A. fumigatus had the L98H amino acid substitution in combination with a 34-base pair tandem repeat in the promoter region, one isolate had an M220I mutation, and another itraconazole-resistant isolate did not have a substitution in the cyp51A gene.

CONCLUSIONSThis study shows that triazole-resistant A. fumigatus clinical isolates are present in Nanjing, China, which is a new challenge to the clinical management of IA.

Antifungal Agents ; pharmacology ; Aspergillus fumigatus ; drug effects ; genetics ; China ; Drug Resistance, Fungal ; Itraconazole ; pharmacology ; Microbial Sensitivity Tests ; Promoter Regions, Genetic ; genetics ; Tandem Repeat Sequences ; genetics ; Triazoles ; pharmacology ; Voriconazole ; pharmacology

BACKGROUNDAspergillus fumigatus (A. fumigatus) is a ubiquitous saprophytic fungus responsible for the majority of invasive mold infections in patients undergoing chemotherapy, organ transplantation or with persistent neutropenia. This study aimed to determine the role of E-cadherin for adhesion and endocytosis of A. fumigatus blastospores in the human epithelial cell line A549.

METHODSA. fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure. After establishing the adhesion model, the adhesion and endocytosis of A. fumigatus blastospores by A549 cells were evaluated by down-regulating E-cadherin of A549 cells using blocking antibody or small interfering RNA (siRNA).

RESULTSE-cadherin was adhered to the surface of A. fumigatus blastospore. Adhesion and endocytosis of the blastospores were reduced by blocking or down-regulating E-cadherin in A549 cells.

CONCLUSIONSE-cadherin is a receptor for adhesion and endocytosis of A. fumigatus blastospores in epithelial cells. This may open a new approach to treat this fungal infection.

Aspergillus fumigatus ; cytology ; Cadherins ; genetics ; metabolism ; Cell Line ; Endocytosis ; physiology ; Epithelial Cells ; metabolism ; microbiology ; Fungal Proteins ; chemistry ; metabolism ; Humans ; In Vitro Techniques ; Protein Binding ; physiology ; RNA, Small Interfering ; Spores, Fungal ; cytology

Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.
6-Phytase ; chemistry ; genetics ; metabolism ; Amino Acid Substitution ; Aspergillus fumigatus ; enzymology ; genetics ; Biocatalysis ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; chemistry ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Models, Molecular ; Mutagenesis, Site-Directed ; Mutant Proteins ; genetics ; metabolism ; Mutation ; Pichia ; genetics ; Polymerase Chain Reaction ; Protein Conformation ; Protein Engineering ; methods ; Recombinant Proteins ; metabolism ; Structure-Activity Relationship ; Substrate Specificity