Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach

BACKGROUND: Approximately 20% of the world's asthmatics are suffering from Aspergillus fumigatus (Afu)-induced allergies. The characterization of specific IgE-inducing allergens in allergic aspergillosis patients is fundamental for clinical diagnosis and for immunotherapy. METHODS: Immunoproteomics combined with mass spectrometric analysis was used to identify proteins of third-week culture filtrate (3wcf) potentially responsible for Afu-specific IgE immunoreactivity, using pooled sera from Afu-sensitized asthmatics. Their allergenic potential was also tested against patients with allergic bronchopulmonary aspergillosis (ABPA), by two-dimensional (2-D) gel electrophoresis immunoblotting of 3wcf proteins with individual sera from such patients. This helped us to establish a set of candidate allergens, which could be explored further for diagnostic application in allergic aspergillosis asthmatics including ABPA. RESULTS: Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and/or de novo sequencing by MS/MS analysis of the protein spots from 2-D gels led to the identification of a total of 16 allergens of Afu. Eleven of them are being reported as allergens for the first time and five had been reported earlier. Putative isoforms of the proteins Asp f 13 and chitosanase have been observed for the first time. When studied for reactivity of these proteins among patients with ABPA using their individual sera, these patients exhibited sensitization although the pattern was varying. Taken together, these proteins could thus be considered as potential allergens even among patients with ABPA. Three of these proteins viz. the hypothetical protein (# spot no. 5), extracellular arabinase (# spot no. 6) and chitosanase (# spot no. 11) could be major allergens with specific IgE immunoreactivity with six out of eight patients' sera. CONCLUSIONS: The immunoproteomic approach applied to the analysis of culture filtrate proteins resulted in the identification of several candidate allergens, many of them novel, contributing to the catalogue of Afu allergenic proteins, which would facilitate improved serodiagnosis for allergic aspergillosis. In addition, the immunoreactivity of these proteins observed among the patients with ABPA may be potentially useful for its serodiagnosis and opens up further opportunities for the development of personalized immunotherapeutics for patients with ABPA.     

Putative virulence factors of Aspergillus fumigatus

Various putative virulence factors of Aspergillus fumigatus have been studied over the past decades. A. fumigatus gliotoxin is a potent inhibitor of the mucociliary system. Several fungal metabolites interfere with phagocytosis and opsonization including toxins, 'conidial inhibitory factor', 'A. fumigatus diffusible product' and 'complement inhibitory factor'. A. fumigatus can bind specifically to different host tissues components, whereas toxins give a general and significant immunosuppressive effect on host defences. Circumstantial evidence links the production of elastinolytic proteases with the ability to cause disease. However, none of the reports demonstrates conclusively a decisive role for any of the virulence factors described thus far. It is conceivable that proteolytic enzyme activities such as those expressed by AFAlp are one of a number of factors, each with a minor effect, that combine to facilitate disease progression.     

烟曲霉pbs2基因功能初步探讨

目的 克隆烟曲霉pbs2基因,并构建烟曲霉pbs2基因突变株,了解该基因对烟曲霉形态学影响以及对渗透压、氧化压力物质敏感性的变化.方法 通过基因同源性比对,在烟曲霉基因组找出与酿酒酵母pbs2基因同源的序列,分析其结构并通过PCR扩增烟曲霉pbs2基因连同其上下游各约1.0 kb的DNA片段,将该片段重组到质粒pDHt/SK中形成PA,再通过PCR扩增pyrG作为筛选标记插入到PA的pbs2基因开放阅读框架(ORF)中,构建好pbs2基因敲除载体PB.将敲除载体转化到根瘤农杆菌,再利用ATMT法转化嘧啶营养缺陷株烟曲霉AF293.1,筛选出烟曲霉pbs2基因缺陷株△Pbs2.观察突变株和野生株AF293在基础培养基上的生长速度并测定二者对渗透压、氧化压力物质敏感性的变化.结果 经过SMART软件分析发现烟曲霉pbs2基因编码一种磷酸转移酶.在基础培养基上△pbs2生长速度同野生株间差异无统计学意义(P＞0.05),但在氧化压力和渗透压的信号传导中,△pbs2对氧化压力和渗透压的敏感性较野生株AF293敏感.结论 烟曲霉中pbs2基因不仅参与渗透压的传导,还参与氧化压力的传导,但只参与传导特定的氧化压力.     

Antigen and antibody attachment in ELISA for Aspergillus fumigatus IgG antibodies

Four commercial antigen extracts of Aspergillus fumigatus were evaluated for use in a rapid enzyme-linked immunosorbent assay (ELISA) for anti-A. fumigatus IgG. Initial binding of both somatic and culture filtrate preparations to a polyvinyl chloride solid phase was concentration dependent and increased with incubation time. Antigen binding to the solid phase was reproducible. Binding of A. fumigatus precipitin-positive serum to bound antigen was rapid. All four A. fumigatus antigens demonstrated similar dose-response curves when tested against pooled sera containing a high titre of A. fumigatus antibodies. Detectable activity in precipitin test-negative sera decreased rapidly with dilution. All the antigen preparations were found to be suitable for ELISA procedures and permit the rapid determination of IgG antibodies to A. fumigatus.     

Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei

Ascochyta rabiei produces and accumulates one of the well-known fungal polyketides, 1,8-dihydroxynaphthalene-melanin pigment (DHN-melanin), in asexual and sexual fruiting bodies. Degenerate PCR primers were used to isolate an ArPKS1 of A. rabiei encoding a polypeptide with high similarity to polyketide synthase (PKS) involved in biosynthesis of DHN-melanin in other ascomycetous fungi. Site-directed mutagenesis of ArPKS1 in A. rabiei generated melanin-deficient pycnidial mutants but caused no significant reduction of pathogenicity to chickpea. Pycnidiospores in ArPKS1-mutant pycnidia showed higher sensitivity to UV light exposure compared to pycnidiospores in melanized pycnidia of the wild-type progenitor isolate. Integration of an orthologous PKS1 gene from Bipolaris oryzae into the genome of the mutants complemented the dysfunctional ArPKS1 gene. This study demonstrated that A. rabiei uses a DHN-melanin pathway for pigmentation of pycnidia and this molecule may protect pycnidiospores from UV irradiation.     

Release of antigens and allergens during shake-culture of Aspergillus fumigatus

The appearance of IgG and IgE binding components in the medium of shake cultures of Aspergillus fumigatus has been studied. Cultures were grown in synthetic asparagine medium at 35 degrees C and flasks harvested in duplicate 1, 2, 3, 4, 7 and 14 days after inoculation. The pH of the medium dropped from its initial value of 5.5 to pH 3, and then after 4 days gradually increased up to pH 7.5 in the 14-day medium. The weight of mycelium, after an initial peak followed by a slight decline, increased as the pH of the medium increased. Components able to bind IgG and IgE from pooled ABPA sera were detected by crossed immunoelectrophoresis/self-crossed radioimmunoelectrophoresis within 24 h of growth, but maximal release of both antigens and allergens coincided with the increase in pH of the medium and was seen in the 14-day culture filtrate. Two recognised "major" antigens, Ag 7 and Ag 13, detected using the relevant monospecific antisera, were present in the culture medium after 14 days of growth and similarly for Ag 3, the major allergen, although another allergen, Ag 1, was identified in the 1-day extract. None of the culture filtrates was found to contain the "C-substance" polysaccharide.     

固有免疫在脂多糖、烟曲霉菌诱导的角膜细胞耐受中的作用

研究烟曲霉菌(AF)、脂多糖(LPS)能否引起角膜细胞的耐受反应,增强角膜对病原体刺激的防御功能。小剂量AF、LPS分别预处理永生化人角膜上皮细胞(THCE)和永生化人角膜基质细胞(THSF),20 h后给予大剂量AF或LPS再次刺激。于再次刺激后4 h收集细胞上清,酶联免疫吸附试验(ELISA)检测细胞因子IL-8的分泌,趋化实验检测LPS预处理引起THSF对PMN趋化的改变。于再次刺激后1 h收集细胞,实时RT-PCR检测细胞因子IL-8 mRNA和抗菌肽CCL20和LL-37 mRNA的表达。结果显示AF、LPS分别预处理THCE和THSF均可显著抑制IL-8的分泌。LPS预处理THSF抑制IL-8 mRNA的表达,增加CCL20和LL-37 mRNA的表达,并且抑制中性粒细胞(PMN)向角膜细胞的趋化。因此角膜细胞的耐受反应可避免过度的炎症反应,减轻角膜感染,在角膜炎症中起保护性作用。     

烟曲霉菌感染小鼠肺组织中NOD1信号通路的激活

目的研究天然免疫NOD1信号通路在烟曲霉菌感染小鼠肺组织中是否得以激活。方法小鼠分为正常对照组和感染组,感染组小鼠经鼻滴入烟曲霉菌孢子24 h、48 h、72 h后分别处死,碾碎肺组织平板培养以检测其肺组织烟曲霉菌负荷,观察肺组织病理学改变,RT-PCR检测NOD1及RIP2 mRNA表达量、Western Blot检测胞核NF-κBp65和胞浆TNF-α在小鼠肺组织中的含量。结果①感染组小鼠肺组织48 h时Af负荷达最高且可见严重的充血和出血,有大量的炎症细胞浸润,72 h时Af负荷迅速下降且未见菌丝形成,仅见轻微的充血、出血和炎症反应;正常组烟曲霉菌培养阴性且肺组织结构正常;②RT-PCR及Western Blot结果显示:感染组小鼠肺组织各时相点NOD1 mRNA、RIP2 mRNA、NF-κBp65和TNF-α的表达量均明显高于正常对照组(P<0.05),前三者均在48 h表达最高,TNF-α在72 h表达最高(P<0.05)。结论NOD1信号通路在烟曲霉菌感染的小鼠肺组织中得以活化,其介导的天然免疫对抗烟曲霉菌感染和保护小鼠肺组织起了一定的作用。     

Aspergillus fumigatus corneal infection is regulated by chitin synthases and by neutrophil–derived acidic mammalian chitinase

Aspergillus fumigatus is an important cause of pulmonary and systemic infections in immune compromised individuals, and of corneal ulcers and blindness in immune competent patients. To examine the role of chitin synthases in Aspergillus corneal infection, we analyzed Aspergillus mutants of chitin synthase family 1 and family 2, and found that compared with the parent strain, the quadruple mutants from both families were more readily killed by neutrophils in vitro, and that both also exhibited impaired hyphal growth in the cornea. Further, inhibition of chitin synthases using Nikkomycin Z enhanced neutrophil killing in vitro and in vivo in a murine model of A. fumigatus corneal infection. Acidic mammalian chitinase (AMCase) is mostly produced by macrophages in asthmatic lungs; however, we now demonstrate that neutrophils are a major source of AMCase, which inhibits hyphal growth. In A. fumigatus corneal infection, neutrophils are the major source of AMCase, and addition of AMCase inhibitors or adoptive transfer of neutrophils from AMCase^?/? mice resulted in impaired hyphal killing. Together, these findings identify chitin synthases as important fungal virulence factors and neutrophil‐derived AMCase as an essential mediator of host defense.     

Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures

Abstract

Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8⁺ IL-17⁺ (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia.     

Structure and regulation of cysD, the homocysteine synthase gene of Aspergillus nidulans

The A. nidulans cysD gene encoding homocysteine synthase (O-acetyl-L-homoserine sulphydrylase) has been isolated by functional complementation of a cysD11 mutation. The gene contains five short introns and codes for a protein of 437 amino acids. The protein shows homology with bacterial and yeast O-acetyl- and O-succinyl-homoserine sulphydrylases, particularly from Schizosaccharomyces pombe, Saccharomyces cerevisiae and Kluyveromyces lactis. The cysD cDNA is able to complement a S. cerevisiae mutation impairing homocysteine synthase. Synthesis of the cysD mRNA is down-regulated by a high concentration of methionine in growth medium without sulphate and up-regulated under sulphur limitation. A comparison of cysD genomic and cDNA copies, derived from different A. nidulans strains, revealed a marked DNA-sequence polymorphism manifested mostly by silent point mutations. There was, however, much less polymorphism in the protein sequence. Received: 18 July / 27 October 1997     

Binding of live conidia of Aspergillus fumigatus activates in vitro-generated human Langerhans cells via a lectin of galactomannan specificity

Aspergillus fumigatus is the most common aetiological fungus responsible for human pulmonary aspergilloses. This study investigated the primary contact between Langerhans cells (LC), corresponding to dendritic cells present in pulmonary mucosa and live conidia of A. fumigatus. LC play a key role in antigen presentation for initiation of the primary T cell response. In vitro-generated LC (iLC) were differentiated from cultured human cord blood CD34+ cells and incubated at 4 degrees C or 37 degrees C with fluorescein-isothiocyanate (FITC)-stained conidia or control latex beads. In vitro, conidia were shown by microscopy and cytometry to adhere to iLC in a dose- and time-dependent manner. This adhesion was not limited to iLC because interstitial dendritic and other cells also fluoresced in the presence of conidia-FITC. A lectin other than mannose receptor-type lectin was demonstrated to be responsible of conidial binding. Inhibition of binding was observed with heterologous galactomannan and EDTA, indicating a C-lectin-like receptor with galactomannan structure specificity. After binding only a few conidia were internalized in acidic vesicles, as indicated by the cessation of conidial fluorescence. Conidial binding was followed by activation and maturation of iLC, suggesting that LC present in the lung may play a role in cellular host defence against aspergilloses.     

Endotoxin contamination contributes to the pulmonary inflammatory and functional response to Aspergillus fumigatus extract inhalation in heaves horses

BACKGROUND: As part of a primary prevention of asthma study, we measured the effect of environmental control measures on Der p 1, Fel d 1 and Can f 1 over a 3.5-year period. METHODS: High-risk infants (both parents atopic) without pets, were randomized to the Active group (n = 142, vinyl flooring in child's room, allergen-impermeable cot mattress, hot-washable toy, mite allergen-impermeable encasings to parental bed and to child's bed when older, high filtration vacuum cleaner, hot-washing of bedding) or the Control group (n = 136, no intervention), in early pregnancy. Dust samples from the parental mattress, living room floor, child's mattress and floor at baseline (pregnancy), birth and at 3 years were analysed for Der p 1, Fel d 1 and Can f 1. RESULTS: A total of 278 families completed the baseline visit, 259 the birth visit and 239 the 3-year visit. In the Active group at 3 years, 58% remained compliant with all measures likely to reduce the child's exposure to allergen and 77% of parents still used encasings on their bed. Levels of Der p 1, Fel d 1 and Can f 1 were significantly lower in the Active group in the child's floor and the child's mattress at 3 years compared to the Control group (P < 0.001). For the parental mattress, the levels of Der p 1 and Fel d 1 were lower in the Active group (P < 0.001) and there was a strong trend towards a lower level for Can f 1. There was no difference in the levels of any of the allergens between the groups in the living room floor. Childrens' bedrooms with no detectable mite, cat or dog allergen were significantly more common in the Active than the Control group (25 vs. 2, P < 0.001). CONCLUSIONS: Environmental control measures are effective in substantially reducing levels of Der p 1, Fel d 1 and Can f 1 in homes without pets in the long term and are acceptable to families. The effect of this environmental manipulation on the development of sensitization and allergic disease remains to be seen.